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1
Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-2
lyase 3
4
Se-Young Juna, Steven A. Sattler
b, Gabriel S. Cortez
a, Wilfred Vermerris
c,d, Scott E. Sattler
e, 5
ChulHee Kanga,b
6
7
aDepartment of Chemistry, Washington State University, Pullman, WA 99164 8
bSchool of Molecular Biosciences, Washington State University, Pullman, WA 99163 9
cDepartment of Microbiology & Cell Science - IFAS and
dUF Genetics Institute, University of 10
Florida, Gainesville, FL 32610 11 eU.S. Department of Agriculture – Agricultural Research Service, Wheat, Sorghum and Forage 12
Research Unit, and Department of Agronomy and Horticulture, University of Nebraska, 13
Lincoln, NE 68583 14
15
S.J., W.V., S.E.S., C.K. conceived this project and designed experiments. S.J. performed 16
experiments. S.J., C.K. analyzed data. S.J., W.V., S.E.S., C.K. wrote the article. 17 18
19
This work was supported by the National Science Foundation (grant no. DBI 0959778 to C.K.), 20
the National Institutes of Health (grant no. 1R01GM11125401 to C.K.) and the M.J. Murdock 21
Charitable Trust (to C.K.); by the U.S. Department of Energy’s Office of Energy Efficiency and 22
Renewable Energy, Bioenergy Technologies Office and sponsored by the U.S. DOE’s 23
International Affairs (grant no. DE–PI0000031 to W.V.); by the Biomass Research and 24
Development Initiative (grant no. 2011–1006–30358 to W.V.); and by the U.S. Department of 25
Agriculture (National Institute of Food and Agriculture AFRI grant no. 2011–67009–30026 to 26
S.E.S. and CRIS project grant no. 3042–21220–032–00D). 27
28
ABSTRACT 29
Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway 30
catalyzing the nonoxidative elimination of ammonia from L-phenylalanine to give trans-31
cinnamate. In monocots, PAL also displays tyrosine ammonia lyase activity (TAL), leading to 32
the formation of p-coumaric acid. The catalytic mechanism and substrate-specificity of a major 33
PAL from Sorghum bicolor (SbPAL1), a strategic plant for bioenergy production, were deduced 34
from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and 35
thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a 36
unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) 37
domain compared to that of dicotyledonous plants. The sidechain of His123 in the MIO domain 38
dictated the distance between the catalytic MIO prosthetic group created from 189
Ala-Ser-Gly191
39
residues and the bound L-phenylalanine and L-tyrosine, conferring the deamination reaction 40
Plant Physiology Preview. Published on December 1, 2017, as DOI:10.1104/pp.17.01608
Copyright 2017 by the American Society of Plant Biologists
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2
through either Friedel–Crafts or E2 reaction mechanism. Several recombinant mutant SbPAL1 41
enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 42
6.2 times greater PAL activity without significant TAL activity. This enhancement could 43
establish the basis for further engineering sorghum PALs for potential benefits on both 44
silage/forage quality as well as production of renewable fuels and chemicals from plant biomass. 45
Additional PAL isozymes of sorghum were characterized and categorized into three groups. 46
Taken together, this approach identified critical residues and explained substrate-preference 47
among PAL isozymes in sorghum and other monocots, which can serve as the basis for the 48
engineering of plants with enhanced biomass conversion properties, disease resistance, or 49
nutritional quality. 50
51
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3
INTRODUCTION 52
53 Phenolic metabolism in plants plays important roles in providing aromatic amino acids, defense-54
related compounds, chemical attractants or repellents, structural support, UV-protection, and 55
color (Vermerris and Nicholson, 2006, Lattanzio et al., 2008). The biosynthesis of phenolic 56
compounds in plants occurs via the concerted action of the shikimate and phenylpropanoid 57
pathways, whereby substrates generated from the catabolism of glucose-6-phosphate are 58
converted to chorismate and p-coumaroyl-CoA, respectively (Aharoni and Galili, 2011). 59
Phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) is the first enzyme in the general 60
phenylpropanoid pathway, and significant amount of the total fixed carbon is directed through 61
this enzyme (Maeda and Dudareva, 2012, Zhang and Liu, 2015). PAL catalyzes the non-62
oxidative elimination of ammonia from L-phenylalanine to give trans-cinnamic acid. The 4-63
methylidene-imidazole-5-one (MIO) group is the coenzyme moiety established auto-catalytically 64
by cyclization and dehydration of an Ala-Ser-Gly tripeptide within the sequence of PAL and a 65
closely related enzyme, histidine ammonia lyase (HAL) (Schwede et al., 1999, Langer et al., 66
1997) 67
Following PAL activity, the hydroxylation catalyzed by cinnamate 4-hydroxylase (C4H) gives 68
rise to p-coumaric acid (Russell, 1971). Both trans-cinnamic acid and p-coumaric acid are 69
precursors of myriads of organic compounds with agricultural, nutritional, and industrial 70
relevance, such as stilbenes, chalcones, flavonoids, cinnamoyl anthranilates, monolignols, 71
lignans and lignin (Cheynier et al., 2013, Schreiner et al., 2012, Shahidi and Ambigaipalan, 72
2015, Treutter, 2006, Laskar et al., 2010, Vanholme et al., 2010). Thus, control of enzymatic 73
activity of PAL and C4H can influence the pool of precursors and their fate, making these two 74
enzymes attractive targets for the engineering of plants with enhanced biomass conversion, 75
disease resistance and/or nutritional quality. 76
Reduction of PAL and C4H activities in tobacco through an antisense repression resulted in both 77
reduced content and altered subunit composition of lignin (Sewalt et al., 1997). In addition, PAL 78
and C4H in tobacco were shown to co-localize to the endoplasmic reticulum (ER) membrane 79
(Achnine et al., 2004). Thus, this physical association of PAL with C4H might establish a 80
metabolic channeling complex on the ER surface, through which intermediates can be processed 81
without unnecessary diffusion into the cytosol. This implies that modification of interaction 82
between PAL and C4H could have a much greater impact on metabolic flux than based purely on 83
their catalytic mechanism. If the same is true among strategic grasses for bioenergy production 84
such as sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum), in-depth knowledge 85
about interaction between PAL and C4H together with a detailed understanding of the substrate-86
binding pockets of PAL and C4H will form the basis for engineering altered carbon-flux. 87
While it is known that PAL enzymes in dicots utilize only L-phenylalanine as substrate, PAL 88
from some monocots, including maize (Zea mays; (Rösler et al., 1997) and brachypodium 89
(Brachypodium distachyon; (Cass et al., 2015, Barros et al., 2016) can also deaminate L-tyrosine, 90
indicating their additional activity of tyrosine ammonia-lyase (TAL). While a His residue at 91
position 123 appears to be critical for TAL activity (Röther et al., 2002; Watts et al., 2006; Hsieh 92
et al., 2010), the biochemical basis and evolutionary benefit of TAL activity, which can bypass 93
C4H in the production of trans-p-coumaric acid, are not understood well. Rösler et al (1997) 94
speculated TAL activity might provide metabolic flexibility when phenylalanine concentrations 95
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4
are temporarily low due to reduced biosynthesis, incorporation in proteins, or sequestration in the 96
vacuole. (Maeda, 2016) suggested that PAL activity remained under selective pressure even 97
though generating p-coumarate via TAL activity is energetically more efficient, because of the 98
importance of cinnamic acid as a precursor for defense-related compounds. The importance of 99
TAL activity in the biosynthesis of lignin in brachypodium was demonstrated by transgenic 100
down-regulation of both BdPAL1 and BdPAL2 (Cass et al., 2015) or just BdPAL1 (Borras et al., 101
2016) via RNAi. In both studies, lignin in the transgenic plants displayed a greater syringyl-to-102
guaiacyl ratio, consistent with the phenotypes observed in transgenic tobacco (Sewalt et al., 103
1997) and arabidopsis pal1/pal2 double mutants (Rohde et al., 2004). The change in S/G ratio 104
observed in these different species, combined with gene expression analyses (Rohde et al., 2004; 105
Cass et al., 2015; Borras et al., 2016) suggests the existence of complex regulatory mechanisms 106
that alter flux through the different branches of the general phenylpropanoid pathways. Tracer 107
studies in brachypodium with radiolabeled substrates indicated that close to half of the lignin 108
susceptible to thioacidolysis originated from precursors generated from TAL activity (Borras et 109
al., 2016). 110
The improved saccharification efficiency of biomass from transgenic brachypodium plants in 111
which BdPAL1 and BdPAL2 were down-regulated (Cass et al., 2015) provides evidence that 112
modifying PAL activity can enhance biomass conversion. Given that down-regulation of 113
orthologs encoding monolignol biosynthetic genes in different grass species generates similar 114
phenotypes (e.g. maize and sorghum mutants with reduced caffeic acid O-methyltransferase 115
activity; Vermerris et al., 2007), it is plausible that modification of PAL activity will lead to 116
improved biomass conversion in other grasses. Sorghum has been proposed as a strategic high-117
yielding biomass crop in the U.S., because it is a C4-species with substantial heat and drought 118
tolerance. Its sequenced genome facilitates genetic improvement (Sarath et al., 2008, Paterson et 119
al., 2009). Analysis of several sorghum brown midrib (bmr) mutants, which contain brown 120
vascular tissue in the leaves and stems as a result of perturbations in the monolignol biosynthetic 121
pathway, has shown that their biomass is more easily converted than their wild-type counterparts 122
(Oliver et al., 2005, Jung et al., 2012, Dien et al., 2009, Saballos et al., 2008, Sattler et al., 2010, 123
Sattler et al., 2012, Vermerris et al., 2007) without major negative impacts on agronomic 124
performance (Oliver et al., 2005), indicating that it is possible to balance changes in cell wall 125
composition with plant productivity. The use of sorghum bmr mutants can also reduce the 126
severity of thermo-chemical pretreatment, reducing both the cost of processing and the 127
degradation of monomeric sugars (Dien et al., 2009, Godin et al., 2016). There are no known 128
sorghum bmr mutants with a defective PAL gene. This is not entirely surprising since the 129
sorghum genome contains eight PAL genes (Xu et al., 2009), similar to the observed numbers in 130
maize (10), rice (9) (Penning et al., 2009) and brachypodium (8; Cass et al., 2015). Of the eight 131
sorghum PAL genes, SbPAL1 is the most highly expressed (Shakoor et al., 2014). PAL 132
expression appears to be sensitive to perturbations in the biosynthesis of monolignols, based on 133
semi-quantitative RT–PCR analysis of thirteen sorghum bmr mutants (Yan et al., 2012). 134
Here, we report a comprehensive characterization of SbPAL1 including differential activity for 135
L-tyrosine vs. L-phenylalanine. The key residues that are responsible for PAL/TAL activity were 136
delineated through the high-resolution crystal structure of apo-form SbPAL1, which is the first 137
PAL structure from a monocot, followed by validation via site-directed mutagenesis. 138
139
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5
RESULTS 140
Oligomeric state and global structure of SbPAL1 141
Recombinant Sorghum bicolor PAL1 enzyme (SbPAL1, 75.6 kDa) was purified and crystallized 142
in a tetragonal space group I4122, and its structure was determined at 2.5Å resolution (Table 1). 143
The lattice packing of SbPAL1 was established through a homodimer as an asymmetric unit, 144
which in turn formed a tight interaction with a neighboring homodimer in a two-fold symmetry 145
manner, indicating its homotetrameric nature with a pseudo D2 symmetry (Fig. 1A) as similar to 146
other related ammonia-lyases and ammonia-transferases (Wang et al., 2008, Moffitt et al., 2007, 147
Calabrese et al., 2004, Wang et al., 2005, Ritter and Schulz, 2004, Heberling et al., 2015, Louie 148
et al., 2006). In order to investigate the plausible oligomeric state of SbPAL1 in solution, the 149
PISA software package (Krissinel and Henrick, 2007) was applied to evaluate interactions 150
between neighboring molecules in crystal lattices for predicting biologically relevant oligomeric 151
states. The results clearly showed that SbPAL1 will form a stable homotetramer as indicated in 152
its crystal lattice, where the solvation free energy gain upon formation of the interface (ΔGint) 153
was estimated as -142.3 kcal/mol. Upon tetramerization, a solvent-exposed surface area of this 154
tetrameric SbPAL1 was predicted to be 84,010 Å2 and the area buried due to tetramerization was 155
36,110 Å2. 156
Among 704 residues of SbPAL1, the electron density for the first nine residues of subunit A and 157
three N-terminal residues and the residues 231-240 of subunit B in the dimeric asymmetric unit, 158
respectively, and the last two C-terminal residues of both subunits were not resolved probably 159
due to their disordered nature. The Cα positions of the individual SbPAL1 subunits were 160
superimposable with a root mean square deviation (rmsd) value of 0.65 Å. The B-factor values 161
of the Cα atom indicate that three regions of SbPAL1, residues 90-110, 310-330 and 520-650, 162
display high mobility. The rmsd value between two subunits was reduced to 0.12 Å, without 163
including those three high B-factor regions. 164
Each SbPAL1 subunit contained twenty α-helices and eight short β-strands. Overall, those 165
secondary structural elements established three distinct domains: the MIO, shielding and core 166
domains, which were named in the 3D structure of PAL from parsley (Petroselinum crispum) 167
(Ritter and Schulz, 2004). As shown in Fig. 1B, the residues spanning from Ser10 to Thr249 168
establish a MIO domain (cyan) that contains the 4-methylidene-imidazole-5-one (MIO) 169
prosthetic group and a highly flexible inner lid-loop. From the early stage of refinement, there 170
was clear electron density connected methylidene carbon atom of MIO groups in both subunits 171
and was assigned as NH2 adduct that could be originated from ammonium acetate in the 172
crystallization buffer. This attached ammonium group was surrounded by the sidechains from 173
Leu193, Asn247, Tyr338 and Phe387. The shielding domain of SbPAL1, which is depicted in 174
purple (Fig. 1B), spans from Leu512 to Arg636 and contains four α-helices. According to our 175
search, this shielding domain seems unique to PAL and is absent in both TAL and histidine 176
ammonia lyase (HAL) that have been deposited in Protein Data Bank so far. Generally, the 177
closely related phenylalanine aminomutases (PAM) have a similar sized shielding domain that 178
shares ~27 % amino acid sequence identity with that of PAL. In the tetrameric configuration of 179
SbPAL1, this shielding domain established an arch-like structure over the active site of the MIO 180
domain. Lastly, the core domain, which was depicted in beige (Fig. 1B), connects those catalytic 181
and shielding domains. The longest alpha helix (α19: Gly481 – Gln529), which is located at the 182
center of Fig. 1B with two-thirds in beige color and one-third in purple, runs through the core 183
domain starting right under the stem of the MIO group to the end of the shielding domain. 184
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6
Active site of SbPAL1: In the crystal structure of each SbPAL1 subunit, 189
Ala-Ser-Gly191
185
tripeptide displayed a well-defined the electron density of the MIO motif. Each active site 186
containing this MIO cofactor was established with residues from three neighboring subunits of a 187
homotetramer. For example, the active site of A subunit was constituted not only by the residues 188
of A subunit but also by the residues from B and B’ subunits that are related by 189
noncrystallographic and crystallographic two-fold symmetry respectively. The individual 190
imidazole-5-one ring of the MIO cofactor retained aromaticity where its N3 atom showed a 191
planar sp2 conformation. On the other hand, the N2 and N3 atoms on the MIO ring established a 192
hydrogen bond with the sidechain of Tyr338 and Asn247 respectively. 193
The above-mentioned two flexible loops near the active site have been referred previously as 194
inner lid-loop and outer lid-loop (Louie et al., 2006). The inner lid-loop of SbPAL1 (residues 90-195
110) caps the active site, and the outer lid-loop of SbPAL1 (residues 310-330) flanks the inner 196
lid-loop of the dyad-related subunit. It has been known that the conformations of these two loops 197
vary significantly among aromatic ammonia lyases. For example, the corresponding outer lid-198
loop of RsTAL from Rhodobacter sphaeroides (Louie et al., 2006) is longer than that of SbPAL1 199
or parsley PcPAL (Ritter and Schulz, 2004). The longer outer lid-loop of RsTAL seems pressing 200
down the capping inner lid-loop and thus results in a tighter closure for the active site pocket. 201
Due to the extra tight closure, the Tyr60 side chain of RsTAL is positioned closer to the 202
substrate, providing the second proton acceptor for the E2-type reaction for L-tyrosine 203
deamination catalysis (Louie et al., 2006). As PAL catalyzes a Friedel-Crafts-type reaction for 204
deamination of L-phenylalanine (Hermes et al., 1985 , Schuster and Rétey, 1995, Louie et al., 205
2006, Alunni et al., 2003, Watts et al., 2006, Pilbák et al., 2012), PAL does not require the 206
second proton acceptor. Thus, the outer lid-loop is generally shorter in their structures. The 207
shielding domain of SbPAL1, established by the residues 520-650, is not present among TALs 208
and HALs that have been characterized so far. Although the exact role of this domain unique to 209
PAL is unknown, it could play a similar role as the outer lid-loop. 210
211
The substrate docking and identification of key residues for binding and catalysis 212
Despite our numerous attempts, complex crystals of SbPAL1 with either substrate analogs or 213
product were not obtained. Therefore, in order to understand the potential mode of 214
substrate/product-binding, an approach with molecular docking software was adopted through 215
AutoDock Vina (Trott and Olson, 2010). Although a complex crystal structure for PAL has not 216
been obtained so far, our docked position and conformation are very similar to those bound 217
ligands in the structures of RsTAL (Louie et al., 2006) and 2,3-aminomutase from Streptomyces 218
globisporus (Bruner and Cooke, 2010). The docking result clearly indicated that the substrate-219
binding pocket of SbPAL1 was formed with mainly hydrophobic residues. Ionic interaction was 220
noticed between the guanidinium side chain of Arg341 from subunit B (depicted with green in 221
Fig. 2) and the carboxyl group of the L-phenylalanine and L-tyrosine docked in subunit A. In 222
addition, for proper positioning of the aromatic portion of the both bound substrates, the 223
sidechain of Lys443 in subunit B′ and aromatic ring of both substrates docked in subunit A were 224
within an appropriate distance establishing a potential cation-π interaction. The phenolic 225
sidechain of Tyr338 from subunit B was located on top of the Cβ of docked phenylalanine, thus 226
being located at the potential position for a general base during catalysis. In addition, the 227
hydroxyl group of Tyr96 sidechain from the inner lid-loop was positioned near the Cγ of the 228
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7
docked L-phenylalanine. On the contrary, in the binding conformation of L-tyrosine molecule, 229
the hydroxyl group of the same Tyr96 was closer to its Cβ instead. Thus, it is likely that Tyr96 230
serves as one of the two required bases along with Tyr338 for E2 type deamination of L-tyrosine. 231
Another noticeable difference between the docked conformation of L-phenylalanine and L-232
tyrosine was observed. The hydroxyl group of the L-tyrosine substrate interacts with the 233
imidazole sidechain of His123, which consequently shifts the MIO group closer to Cα of the 234
bound L-tyrosine. Absence of this interaction between the imidazole sidechain of His123 and the 235
bound L-phenylalanine positioned the ortho-carbon (C2) of its phenyl ring closer to the MIO 236
group and the amine group closer to the sidechain of Asn474. In order to confirm the position 237
and resulting interactions of bound substrate, the same molecular docking was performed with 238
the model coordinates for H123F-SbPAL1 mutant. The results confirmed that the loss of polarity 239
in the H123F mutant oriented both L-tyrosine and L-phenylalanine in similar positions, where the 240
ortho-carbon is closer to the methylidene carbon of MIO (Fig. 3). 241
242
Steady state kinetics of wild-type and mutant SbPAL1, and isozymes 243
To confirm our hypothesis based on the crystal structure and molecular docking results, enzyme 244
kinetic assays with the wild-type SbPAL1 and its site-directed mutants were performed. It is 245
known that PAL loses activity fast without any reducing agents and that dithiothreitol (DTT) also 246
forms a covalent adduct to the MIO-N atom of PAL (Ritter and Schulz, 2004). Thus, β-247
mercaptoethanol (βME) was tested for its inhibitory effect. In the absence of βME, kcat and KM 248
for phenylalanine deamination were 1.76 s-1
and 0.34 mM, respectively. However, SbPAL1 249
activity was inhibited by βME competitively, where kcat was unaffected but KM was increased as 250
the concentration of βME was increased (Fig. 4). Activity of SbPAL1 was also inhibited by tris 251
2-carboxyethylphosphine (TCEP) (Supplemental Data). Due to these inhibitory effects of 252
reducing agents, all of our enzyme purification and kinetics measurements were performed 253
within 24 hours after harvesting the cells and in absence of any reducing agents. 254
The deamination activity of SbPAL1 against four potential substrates, L-phenylalanine, L-255
tyrosine, L-histidine and L-3,4-dihydroxyphenylalanine (L-DOPA), was assayed (Table 2). Wild-256
type SbPAL1 showed catalytic activity against both L-phenylalanine and L-tyrosine with kcat/KM 257
values of 5.18 s-1
mM-1
and 2.52 s-1
mM-1
, respectively. SbPAL1 displayed activity against L-258
3,4-dihydroxyphenylalanine (L-DOPA) with a catalytic efficiency (kcat/Km) of 0.76 s-1
mM-1
, 259
which is only 14.6% of the activity displayed against L-phenylalanine. SbPAL1 showed no 260
detectable catalytic activity against L-histidine. 261
To confirm the effect of above-mentioned His123, enzyme kinetics of H123F- and H123Y-262
SbPAL1 mutants were tested. The H123F-SbPAL1 mutant showed 6.2-fold elevation in catalytic 263
efficiency (kcat/KM) for L-phenylalanine, mainly due to a 5.7-fold decrease in KM. However, 264
H123F-SbPAL1 lost activity for L-tyrosine, and thus became a dedicated PAL. For the H123Y-265
SbPAL1 mutant, the catalytic efficiency for L-phenylalanine was reduced to 0.72 s-1
mM-1
, 266
mainly due to a 10-fold decrease in the turnover rate (kcat). The H123Y-SbPAL1 mutant also lost 267
activity against tyrosine. Neither the H123Y- nor the H123Y-SbPAL1 mutants showed any 268
activity with L-histidine (Table 2, Fig. 4B). 269
To further confirm a hypothetical role of Phe/His123 in substrate preference, two additional PAL 270
isozymes, encoded by Sorghum bicolor genes Sb06g022740 and Sb04g026520, were tested. At 271
the corresponding position of His123 of SbPAL1, Sb06g022740 has a histidine and 272
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8
Sb04g026520 has a phenylalanine. Consistent with the kinetics data of SbPAL1 and the H123Y-273
SbPAL1 mutant, Sb06g022740 catalyzed deamination of both L-phenylalanine and L-tyrosine 274
with kcat values of 2.34 s-1
and 0.37 s-1
, respectively. In contrast, Sb04g026520 only catalyzed 275
deamination of L-phenylalanine effectively, with a kcat of 2.20 s-1
(Table 2, Fig. 4B). 276
Close examination of the crystal structure of SbPAL1 also suggested that the spatial position of 277
residue Phe102 is close to and almost parallel to the sidechain of Tyr96 (Fig. 2) and that an 278
F102Y mutation may provide an alternative catalytic base when the flexible inner lid-loop is in 279
motion. Because the sidechain of Tyr96 serves as the second catalytic base during deamination 280
reaction of L-tyrosine, an F102Y mutation might increase catalytic activity of SbPAL1 for L-281
tyrosine. To test this hypothesis, the enzyme kinetics of an F102Y-SbPAL1 mutant were 282
examined. As predicted, the turnover rate for L-tyrosine was improved 3.6-fold, whereas the 283
turnover rate for L-phenylalanine was unaffected. However, KM for both L-tyrosine and L-284
phenylalanine were increased by 7.6-, and 24-fold, respectively, which caused reduced enzyme 285
efficiency for both substrates. The F102Y mutant did not show any activity with L-histidine 286
(Table 2, Fig. 4B). 287
To examine the hypothetical role of residue Lys443, the enzyme kinetics of a K443E-SbPAL1 288
mutant were tested. This mutant showed no measurable activity against any of the three 289
substrates, L- phenylalanine, L-tyrosine, and L-histidine (Table 2, Fig. 4B), consistent with our 290
prediction about its role for proper positioning of the aromatic portion of the bound substrates 291
through a potential cation-π interaction. 292
According to the proposed mechanism, residue Tyr96 is one of the two bases involved in an E2-293
type deamination of L-tyrosine, but is not required for a Friedel-Crafts-type deamination of L-294
phenylalanine (Fig. 8). To examine whether SbPAL1 uses two different mechanisms, activity of 295
Y96F-SbPAL1 mutant was tested for both substrates. However, Y96F-SbPAL1 mutant displayed 296
only a trace amount of activity for both L-tyrosine and L-phenylalanine (Table 2, Fig. 4B). 297
Subsequent isothermal titration calorimetry experiment also showed no measurable affinity 298
between the Y96F-SbPAL1 mutant and the reaction products, trans-cinnamic acid and p-299
coumaric acid, which reflects significant perturbation of the local conformation. Consistent with 300
that, the equivalent mutation (Y110F) in PcPAL resulted in an almost inactivated enzyme 301
(Röther et al., 2002). 302
Isothermal titration calorimetry (ITC) for wild-type SbPAL1 303
Isothermal titration calorimetry (ITC) was performed to determine the thermodynamic 304
parameters for the products and the substrate-analogs. As shown in Fig. 5A, a large amount of 305
heat was released (H = -2.3 kcal mol-1
) when trans-cinnamic acid, the deamination product of 306
L-phenylalanine, was used as the titrant into the SbPAL1 solution. This enthalpic change was 307
also accompanied by a substantial entropic contribution upon ligand-binding (S = 12.6 cal mol-
308 1 K
-1), resulting in a Kd of 34.1 μM (Fig. 5A). On the other hand, p-coumaric acid, a product 309
formed from deamination of L-tyrosine, bound to SbPAL1 with a Kd value of 2.14 μM with ΔH 310
of -9.1 kcal mol-1
and ΔS of 6.29 cal mol-1
K-1
. A higher affinity of SbPAL1 for p-coumaric acid 311
than for trans-cinnamic acid is likely due to the hydrogen bond formation between the p-312
hydroxyl group of p-coumaric acid and the nearby imidazole sidechain of His123 (Figure 3B). In 313
addition, the Kd value for caffeic acid, a product formed from deamination of L-DOPA was 6.21 314
μM with ΔH of -2.1 kcal mol-1
and ΔS of 16.7 cal mol-1
K-1
. According to the ITC results, 315
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9
SbPAL1 did not show any cooperative binding to either of the products, nor display any 316
significant affinity for either D-phenylalanine or D-tyrosine. 317
Our inhibition assays with 2-aminoindan-2-phosphonic acid (AIP) indicated its minor 318
competitive inhibition of SbPAL1 activity. The Ki value for AIP, estimated from enzyme 319
kinetics, was 0.22 mM, which was near the detection limit of our calorimeter. Being consistent 320
with this result, the Kd value between SbPAL1 and AIP from ITC measurement was 321
insignificant. Low affinity of SbPAL1 to AIP was somewhat expected, as AIP is a PAL-specific 322
inhibitor. SbPAL1 has both PAL and TAL activities (Table 2), and the conformation of its 323
active-site has characteristics of both PAL and TAL (Fig. 3). In supporting these, H123F-324
SbPAL1 showed higher affinity to AIP with a Kd value of 0.26 μM with ΔH of -3.2 kcal mol-1
325
and ΔS of 19.3 cal mol-1
K-1
(Fig. 5B, 5C). 326
327
Structural homologs of SbPAL1 328
To identify homologs of SbPAL1 and correlate their sequences with known substrate-329
specificities, the amino acid sequence of SbPAL1 was used to perform a similarity search with 330
the deposited crystal structures in the Protein Data Bank (PDB) using BLAST (Altschul et al., 331
1997). In addition, a chain fold search using DALI (Holm and Sander, 1993) was performed 332
using the atomic coordinates of SbPAL1 to identify closely-related structural homologs in the 333
same PDB. A BLAST search to identify proteins with similar amino acid sequences in the PDB 334
revealed that PAL from Petroselinum crispum (parsley) (PDBID: 1W27) showed the highest 335
identity (69%) to SbPAL1, followed by phenylalanine-2,3-aminomutase (PAM, PDB ID: 4C6G) 336
from Taxus chinensis (Chinese yew) with 45% identity, PAL from the yeast Rhodosporidium 337
toruloides (PDB ID: 1Y2M) and the cyanobacterium Nostoc punctiforme (PDB ID: 2NYF) with 338
substantially lower values of 36 and 35% identity, respectively. Overall, DALI search results 339
were similar to those of BLAST searches. PAL from Petroselinum crispum was again the most 340
similar 3D structure with a Z-score of 53.9, followed by PAM from Taxus chinensis (Z=49.5), 341
TAM from Streptomyces globisporus (PDBID: 2RJR, Z=40.9), PAL from Anabaena variabilis 342
(2NYN, Z=40.6), PALs from Rhodosporidium toruloides (1Y2M, Z=38.7) and Nostoc 343
punctiforme (2NYF, Z=40.4), PAM from the bacterium Pantoea agglomerans (PDBID: 3UNV, 344
Z=39.6), TAM from Streptomyces globisporus (2OHY, Z=39.6) and HAL from Pseudomonas 345
putida (1B8F, Z=39.5). As shown in both DALI and BLAST searches, SbPAL1 showed a 346
significant similarity with a group of plant PAMs, displaying 45-46% amino acid sequence 347
identity. Both sequence alignment (Fig. 6) and secondary-structure matching (SSM) of above 348
structures displayed a clear correlation, where all eukaryotic PALs and PAMs contain the 349
shielding domain with a shorter outer lid-loop. On the other hand, TAL, HAL, and prokaryotic 350
PAL/PAM lack the shielding domain, and contain a longer lid-loop instead. Among those, the 351
inner lid-loop of PcPAL was significantly different from the others and was referred to as an 352
open active-site form (Louie et al., 2006); Heberling et al., 2015). However, due to its poor 353
fitting into electron density, the conformation of the inner lid-loop in the deposited PcPAL 354
structure is uncertain. 355
356
Sequence comparison and activity assay for the putative SbPAL isozymes 357
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10
In addition to SbPAL1, there are seven other genes encoding putative PAL isozymes in the 358
genome of Sorghum bicolor. According to the alignments of the deduced amino acid sequences 359
(Fig. 7A) and phylogenetic analyses (Fig. 7B), those eight isozymes can be categorized into three 360
groups. The first group contains Sb04g026510 (SbPAL1) and Sb06g022740. The second group 361
contains Sb04g026520, Sb04g026560, Sb04g026550, Sb04g026530, and Sb04g026540. The 362
remaining Sb01g014020 is distinct from those in the other two groups. Significantly, the first 363
group has His at the earlier mentioned residue 123, the second group has Phe, and Sb01g014020 364
has Tyr. In addition, all isozymes have Lys at residue 443 except Sb01g014020, which has Asn. 365
Expression data in Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Sbicolor) 366
indicate that gene Sb01g014020 is only expressed at very low levels in roots. Based on its 367
expression profile, the protein encoded by this gene is not expected to play a significant role in 368
phenylpropanoid metabolism. However, to understand the effect of Tyr in Sb01g014020 at the 369
corresponding position of H123 in SbPAL1, enzyme kinetics of a H123Y-SbPAL1 mutant were 370
examined. H123Y-SbPAL1 was inactive under the test conditions for all three tested substrates; 371
L-phenylalanine, L-tyrosine, and L-histidine (Table 2). Our kinetics results show that the first 372
group, SbPAL1 and Sb06g022740, which have His at residue 123, are bifunctional. In contrast, 373
Sb04g026520, which has Phe at residue 123, displayed only PAL activity, similar to what was 374
observed for the H123F-SbPAL1 mutant (Table 2). 375
376
DISCUSSION 377
The overall structure of SbPAL1 was predominantly α-helical, where 52% of its residues are in 378
23 α-helices. PAL from Petroselinum crispum (PcPAL), which is the most similar in terms of 379
both amino acid sequence and 3D-structure according to our BLAST and DALI searches, shares 380
amino acid sequence with 70% identity with SbPAL1. PcPAL is, so far, the only plant PAL for 381
which a crystal structure is available in PDB. PAL from dicots such as PcPAL are, however, 382
only able to use L-phenylalanine as a substrate, and lack the ability to catalyze the deamination of 383
L-tyrosine. In contrast, several tested monocot PAL enzymes, including SbPAL1, are able to 384
catalyze the deamination of both L-phenylalanine and L-tyrosine. Thus, the TAL activity of 385
SbPAL1 and similar enzymes in other grasses provides an alternative route for generating p-386
coumaric acid without a need for C4H. The catalytic efficiency (kcat/KM) for TAL activity of 387
SbPAL1 is, however, two-fold lower than that of its PAL activity (Table 2). On the other hand, 388
the catalytic efficiency for TAL of Sb06g022740, another bifunctional PAL in lignified tissues, 389
is 1.5 fold higher than PAL activity, although its TAL and PAL efficiencies are 35% and 11% 390
SbPAL1, respectively (Table 2). Borras et al. (2016) determined that BdPAL1 had a two-fold 391
greater activity against L-tyrosine than L-phenylalanine, and that close to half of the lignin 392
susceptible to degradation via thioacidolysis originated from monolignols that could be traced 393
back to TAL activity. Enzymatic properties of ZmPAL1 determined by Rösler et al. (1997) 394
suggest that maize has similar activity towards both substrates and is thus intermediate between 395
the sorghum and brachypodium. The combined results from these three species, taken together 396
with the observation that different grass species have different numbers of PAL homologs 397
encoding enzymes with both PAL and TAL activity (Borras et al, 2016), implies an inherent 398
variation among individual grass species in the regulation of metabolic flux through the general 399
phenylpropanoid pathway and metabolic pathways leading to specific classes of 400
phenylpropanoids. These differences may reflect adaptation of the individual grass species to the 401
specific environments where they evolved, which differ in terms of climate (temperature water, 402
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11
photoperiod, light intensity), pests, and pathogens. This variation in selective pressure may 403
explain the observed variation in photosynthesis (C3 vs. different forms of C4; Williams et al., 404
2013) and the ability to produce certain metabolites, including phenylpropanoids. For example, 405
despite a close evolutionary history, sorghum is able to produce the antifungal 3-406
deoxyanthocyanidins, whereas maize is not (Snyder and Nicholson, 1990). 407
408
Active site and reaction mechanism 409
The diffusing-in substrate docks the active site through replacing water molecules bound in the 410
active site as indicated in our product ITC data (S = 12.6 and 6.29 cal mol-1
K-1
). In the active 411
site, both L-phenylalanine and L-tyrosine establish a salt-bridge between their respective α-412
carboxyl group and the guanidinium sidechain of Arg341 and π-cation interaction between their 413
aromatic ring and the amine sidechain of Lys443. However, L-tyrosine seems to be able to form 414
a tighter bonding than L-phenylalanine through an additional hydrogen bond with the imidazole 415
side chain of His123. On the other hand, L-phenylalanine interacts with the sidechain of Asn474. 416
Obviously, the flexible inner lid-loop must be displaced for substrate to enter, of which length 417
and flexibility will affect the KM and kcat as suggested before (Wang et al., 2008, Ritter and 418
Schulz, 2004, Pilbák et al., 2006). In addition, phosphorylation of the hydroxyl sidechain of 419
Thr536 in SbPAL1 could affect this binding and subsequent catalytic events, as proposed for 420
PAL enzymes from Phaseolus vulgaris (Allwood et al., 1999) and parsley (Ritter and Schulz, 421
2004). As indicated previously in the structure of Rhodosporidium toruloides RtPAL (Calabrese 422
et al., 2004), the prosthetic MIO of SbPAL1 is also under direct influence of the positive poles of 423
three helices, which form a triple coiled coil motif, possibly increasing electrophilicity of MIO 424
(Fig. 1). Due to the existence of a hydroxyl group in L-tyrosine and its consequent binding mode, 425
SbPAL1 appears to catalyze deamination of L-tyrosine and L-phenylalanine differently. L-426
Phenylalanine in SbPAL1 seems to undergo a Friedel-Crafts-type deamination, as suggested 427
before (Schuster and Rétey, 1995, Hermes et al., 1985 , Louie et al., 2006, Alunni et al., 2003, 428
Watts et al., 2006, Pilbák et al., 2012), where the ortho-carbon of the aromatic ring of L-429
phenylalanine forms a covalent bond with the electrophilic methylidene carbon. Once that bond 430
is formed, the hydroxyl group of Tyr338, which is within 2.96 Å from the methylidene carbon 431
and 2.84 Å from the N2 atom of the MIO, deprotonates Cβ of the substrate. While the phenolic 432
side chain of Tyr338 acts as a general base, its protonation can be stabilized by the hydrogen 433
bond with the N2 atom of the MIO and the amide sidechain of Gln475. Then, as the bond 434
between the MIO and the intermediate dissociates, the amine-Cα bond of the intermediate breaks, 435
producing cinnamate and an ammonium ion (Fig. 8A). On the other hand, the phenolic sidechain 436
of Tyr338 deprotonates to the amine group of the bound L-tyrosine before it establishes the N-437
MIO intermediate. Then, an E2 elimination step is catalyzed by deprotonation of the intermediate 438
Cβ by the sidechain of Tyr96, producing p-coumarate. The hydroxyl group on this Tyr96 is 439
within a hydrogen bond distance from the backbone amide nitrogen of Gly103 and nearby water 440
molecules, as in the case of RsTAL (Louie et al., 2006). Thus, it is plausible that the hydroxyl 441
group of Tyr96 is connected to a proton network and has reduced pKa, relaying a proton back 442
and forth from the bulky solvent. The amine group is subsequently released from MIO (Fig. 8B). 443
In this reaction, His123 of SbPAL1 provides a differential positioning of the two substrates, L-444
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12
phenylalanine and L-tyrosine, which was identified previously in bacterial PAL (Louie et al., 445
2006, Moffitt et al., 2007) and confirmed by our kinetic and molecular docking assays. 446
447
Substrate-specificity and PAL isozymes in Sorghum bicolor 448
As suggested by previous studies (Watts et al., 2006) and our results, Phe/His at residue 123 449
serves as the factor determining PAL or TAL activity, with Phe123 stipulating PAL activity and 450
His123 TAL activity. As our molecular docking results indicated, an apparent establishment of a 451
hydrogen bond between the nitrogen atom on the His123 sidechain and the hydroxyl group of the 452
L-tyrosine substrate might orient its amine closer to the methylidene carbon of the MIO group, 453
leading to an N-MIO intermediate followed by an E2-like transition state. Quantum 454
mechanics/molecular mechanics calculations in RsTAL determined that the hypothetical Friedel-455
Crafts (FC) route for ammonia elimination from L-tyrosine was less likely due to the high energy 456
of Friedel-Craft type intermediates (Pilbák et al., 2012). 457
Without this hydrogen bond, as is the case in the H123F-SbPAL1 mutant, the ortho-carbon of 458
the phenyl ring is located closer to the MIO, leading to a deamination reaction of the Friedel-459
Crafts type. Our enzyme kinetic data indicate that SbPAL1 effectively deaminates both L-460
phenylalanine and L-tyrosine, but PAL activity displaying higher catalytic efficiency. The 461
H123F-SbPAL1 mutant displayed only PAL activity. Consistent with these observations, the 462
monocotyledonous ZmPAL, BdPAL1 and SbPAL1, all of which have both PAL and TAL 463
activities, contain histidine at this position, whereas dicotyledonous PALs such as the thoroughly 464
investigated PcPAL, have Phe and display only PAL activity. Changing the corresponding 465
residue from Phe to His, however, causes PALs to gain TAL activity without complete loss of 466
PAL activity (Watts et al., 2006, Röther et al., 2002), whereas changing the corresponding 467
residue from His to Phe causes RsTAL to use L-phenylalanine as its preferred substrate (Louie et 468
al., 2006). Thus, there could be additional residues besides Phe/His123 responsible for the 469
substrate specificity as indicated in PALs from Bambusa oldhamii (Hsieh et al., 2010 ). 470
Sequence alignment of PAL, PAM, TAL, and HAL of various organisms shows that Lys443 is 471
also conserved in L-phenylalanine-specific enzymes (PAL and PAM). However, TAL and HAL 472
have Met at the corresponding position. Our docking results show that Lys or Met at residue 443 473
could form cation-π or sulfur-π interactions, respectively, with the aromatic ring of the bound 474
substrate. To determine this plausible effect of Lys443, enzyme kinetics of the K443E-SbPAL1 475
mutant were examined. As expected, the K443E mutant became inactive for both L-476
phenylalanine and L-tyrosine, probably due to electrostatic repulsion between the anionic 477
carboxylate and electron-rich π-system. Thus, Lys443 seems to be critical for binding and 478
positioning of the substrate aromatic moiety. 479
A similar phenomenon was observed with the two assayed SbPAL isozymes (Table 2). As 480
expected, Sb06g022740, which has His at the residue 123, exhibited PAL and TAL activity, 481
whereas Sb04g026520, which has Phe at this position, displayed only PAL activity. In addition, 482
the turnover rate (kcat) values of both isozymes were comparable to those values of wild-type and 483
H123F-SbPAL1. However, both Sb06g022740 and Sb04g026520 showed significantly 484
decreased substrate-affinity. The KM values of Sb06g022740 were 12-fold and 3.5-fold higher 485
for L-phenylalanine and L-tyrosine, respectively, compared to that of wild-type SbPAL1. 486
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13
Similarly, the KM value of Sb04g026520 for Phe was 25-fold higher than that of H123F-487
SbPAL1. Despite the complete conservation in their constituting residues critical for catalysis 488
and substrate binding (except for Phe/His123), the noticeable differences in KM between 489
SbPAL1 and these two isozymes are significant, meaning Phe/His123 residue is not the only 490
factor that influences the enzyme activity and substrate selectivity. This result also correlates 491
with our aforementioned hypothesis of the long-distance secondary effect onto the catalytic 492
residues, as even small changes in non-critical residues affected binding of the substrate 493
significantly. 494
Another residue that may contribute the differential activity is Phe102, located on the inner lid-495
loop and six residues apart from Tyr96. Due to the folding of the inner lid-loop, the sidechain of 496
the two residues, Tyr96 and Phe102, were 4.46 Å apart from each other. Thus, the sidechain of 497
Phe102 could interact with the bound substrate. As the phenolic sidechain of Tyr96 acts as a 498
general base during the deamination reaction of L-tyrosine, the tyrosine sidechain, as in the 499
F102Y-SbPAL1 mutant, may provide an alternative catalytic base in a deamination reaction for 500
tyrosine leading higher TAL efficiency. Supporting this hypothesis is the observation that the 501
F102Y-SbPAL1 mutant showed an improved kcat for L-tyrosine. Conversely, the unaffected kcat 502
for L-phenylalanine reflects that the Friedel-Crafts type deamination reaction for L-phenylalanine 503
is not impacted by another catalytic base (Tyr102) from the inner lid-loop. However, F102Y-504
SbPAL1 displayed increased KM values for both L-tyrosine and L-phenylalanine. Thus, the 505
F102Y-SbPAL1 mutant has increased turnover rate for L-tyrosine without alteration for that of L-506
phenylalanine, but the efficiency (Kcat/KM) for phenylalanine was diminished. 507
Tyr96 acts as a base in the E2-type deamination of L-tyrosine, but does not have any role during 508
the Friedel-Crafts type deamination of L-phenylalanine. However, Y96F-SbPAL1 mutant 509
showed significant loss of activity for both of the substrates (Table 2, Fig. 4B) and no 510
measurable affinity to either trans-cinnamate or p-coumarate. The area surrounding Tyr96 511
sidechain is hydrophobic due to the presence of Phe102, Leu120, and Leu243. Thus, it is 512
plausible that Tyr96 orients the carboxyl group of substrates in addition to act as a general base 513
for L-tyrosine. To support this notion, Tyr96 is also highly conserved among both ammonia-514
lyases and aminomutases (Fig. 6). 515
516
Conclusion 517
In the U.S., sorghum biomass (stalks and leaves) serves as an important forage crop for livestock. 518
In addition, sorghum is being developed as a bioenergy crop for cellulosic biofuels. Second-519
generation biofuels are produced from monomeric sugars derived from cellulose and 520
hemicellulosic polysaccharides in plant biomass. The presence of lignin makes plant cell walls 521
resistant to breakdown either in livestock digestive systems or in the biomass conversion process 522
that occurs in biorefineries. A key challenge in the development of next-generation bioenergy 523
sorghums is the balance between agro-industrial needs and plant fitness (Casler et al., 2002). 524
While transgenic down-regulation of genes encoding enzymes in monolignol and lignin 525
biosynthesis has been successful in improving biomass conversion (Xu et al., 2011, Jung et al., 526
2013), there is a risk of increased susceptibility to biotic and abiotic stresses. This is illustrated 527
by RNAi-mediated down-regulation of BdPAL1 and BdPAL2 activity in brachypodium. While 528
this led to increased efficiency of enzymatic saccharification of the cell wall polysaccharides, the 529
transgenic plants displayed delayed development and reduced root growth, and became more 530
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14
susceptible to two fungal pathogens. In contrast, tolerance to an insect pest, drought, or UV-531
radiation, was not altered. A more detailed understanding of structure and catalysis of enzymes 532
involved in monolignol and lignin biosynthesis can provide additional tools to tailor cell wall 533
composition (Walker et al., 2013, Walker et al., 2016, Green et al., 2014, Jun et al., 2017, Sattler 534
et al., 2017, Moural et al., 2017), by providing targets for mutations that can be identified in 535
natural or mutagenized populations through forward or reverse genetics approaches (Xin et al., 536
2008, Jiao et al., 2016), or that can be introduced using genome editing tools such as TALEN 537
(Cermak et al., 2011) or CRISPR/Cas9 technology (Jinek et al., 2012, Jiang et al., 2013). 538
The first enzyme of the general phenylpropanoid pathway, PAL, is an attractive target for this 539
approach, given its pivotal role in generating precursors for both lignin and various defense-540
related compounds. Through crystal structure, molecular docking, mutagenesis, kinetic analyses 541
and phylogenetic analyses, we have identified that SbPAL1 and other genes encoding putative 542
PAL isozymes in the genome of Sorghum bicolor can be classified into three groups. The first 543
group, which includes SbPAL1 and Sb06g022740, has both PAL and TAL activity. The second 544
group of five PAL isozymes are dedicated PALs. Sb01g014020, which does not fit in either 545
group, is expressed at low levels in roots, and may not play a significant role in phenylpropanoid 546
metabolism. Changing key features of these enzymes altered their preference for substrate and 547
product. Thus, this study reveals targets for genome editing approaches aimed at tailoring lignin 548
levels in plants to improve conversion biomass into biofuels or forage utilization of livestock 549
without negatively affecting plant growth and responses to biotic and abiotic stresses. 550
While similarities in genome organization and individual sequences among grasses have often 551
enabled results from one grass species to be translated to a related grass species, in the case of 552
PAL, apparent differences in enzyme characteristics between maize, brachypodium and sorghum 553
suggests the existence of species-specific differences that enable optimal performance for the 554
environments these species have adapted to, and that necessitate some caution in extrapolating 555
data from one grass species to another. 556
557
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15
EXPERIMENTAL PROCEDURES 558
559
Chemicals and general 560
Analytical-grade chemicals were obtained from Sigma-Aldrich (St. Louis, MO), Thermo Fisher 561
(Waltham, MA) and Alfa-Aesar (Ward Hill, MA). Screening solutions for crystallization were 562
obtained from Hampton Research (Aliso Viejo, CA). The compound 2-aminoindan-2-563
phosphonic acid (AIP) was generously provided to us by Dr. John Ralph and Dr. Hoon Kim at 564
the University of Wisconsin, Madison. 565
Recombinant enzyme expression and purification 566 The PAL1 cDNA corresponding to Sorghum bicolor gene Sb04g026510 was cloned into pET30a 567
vector for overexpression. For expression of recombinant SbPAL1, 200 mL of Luria–Bertani 568
medium containing 100 µg mL-1
kanamycin and 34 µg mL-1
chloramphenicol were inoculated 569
with a freezer stock of Rosetta cells (EMD Millipore, Bellerica, MA) containing the pET30a-570
SbPAL1 construct and grown overnight at 37 °C while shaking. This culture was used to 571
inoculate 3 L of Luria–Bertani medium, which was grown to an OD600 of 0.4 at 37 °C with 572
shaking. The cells were then brought to 18 °C with continuous shaking, and isopropyl β-thio-573
galactopyranoside was added to a final concentration of 0.2 mM. The culture was grown at 18 °C 574
while shaking for an additional 24 hrs. Cells were collected by centrifugation at 5,000 rpm for 20 575
min at 4 °C. The cell pellet was resuspended in 40 mL lysis buffer (50 mM Sodium phosphate, 576
pH 8.0, 300 mM NaCl and, 15 mM imidazole) and was sonicated five times with 15-s pulses 577
(model 450 sonifier; Branson Ultrasonics, Danbury, CT). The lysate was cleared by 578
centrifugation at 16,000 rpm for 25 min. Cleared supernatant was applied to 15 mL nickel-579
nitrilotriacetate agarose (Qiagen, Germantown, MD), equilibrated with lysis buffer, and placed 580
into a gravity-flow column. The column was washed with 20 column-volumes washing buffer 581
(50 mM Sodium phosphate, pH 8.0, 300 mM NaCl, and 25 mM imidazole), and protein was 582
eluted with elution buffer (50 mM Sodium phosphate, pH 8.0, 300 mM NaCl, and 250 mM 583
imidazole). Column fractions containing SbPAL1 were desalted and concentrated into buffer A 584
(20 mM Tris, pH 8.0, 5% v/v glycerol) using an Amicon 8050 ultrafiltration cell with a 10-kDa 585
cutoff membrane (Millipore). Concentrated protein was applied to a Mono-Q column (GE 586
Healthcare) that was pre-equilibrated with buffer A using a flow rate of 2 mL min-1
. SbPAL1 587
was eluted from the column with a linear NaCl gradient. The fractions containing SbPAL1 were 588
pooled and buffer exchanged into 20 mM Tris, pH 8.0. Reducing agents such as β-589
mercaptoethanol (βME) were not used due to formation of adduct with MIO. All expression and 590
purification of isozymes and mutants were performed in an identical manner to SbPAL1 with 591
SDS-PAGE to check the presence and purity of enzymes after each purification step. Protein 592
concentrations were determined by using BCA assay kit (Thermo Fisher Scientific). 593
594
Site-directed mutations were created in the SbPAL1 coding region by PCR-based amplification 595
using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA). The 596
amplification was performed using complementary plus- and minus-strand oligonucleotides 597
containing the target mutations, and was followed by DpnI (New England Biolabs, Ipswich, MA) 598
digestion to degrade the template prior to transformation of competent E. coli Rosetta cells 599
(EMD Millipore, Billerica, MA). Both mutations were confirmed by DNA sequencing 600
(GENEWIZ, Plainfield, NJ). 601
Crystallization and structure determination 602
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16
Crystals of SbPAL1 were grown through the hanging-drop, vapor-diffusion method. The purified 603
SbPAL1 was concentrated to 10 mg mL-1
in 20 mM Tris pH 8.0, and mixed 1:1 volume with a 604
reservoir solution contained 200 mM Ammonium formate pH 6.6, 20 % w/v polyethylene glycol 605
3,350. Crystals of SbPAL1 appeared within 10 days. Adequate cryo-protection was achieved by 606
passing crystals through a small drop of storage buffer/mother liquor mixture, which was brought 607
to a final concentration of 50 % w/v PEG 3,350. SbCAD4 was crystallized in the space group 608
I4122 and had unit cell dimensions a = b = 126.304 Å, c = 337.477 Å, α = β = γ = 90°. Data were 609
collected to 2.5 Å at the Berkeley Advanced Light Source (ALS; Beamline 5.0.2), with an 610
exposure time of 2 s and a detector distance of 380 mm at 100 K. Diffraction data were scaled 611
using the program HKL2000 (Otwinowski and Minor, 1997). 612
613
Phasing and refinement: Initial phasing of diffraction data was performed by molecular 614
replacement with the Phaser in the PHENIX package (Adams et al., 2010) using the coordinate 615
of PcPAL (PDBI: 1W27) (Ritter and Schulz, 2004) as a search model. The following 616
conformation and position were refined further using PHENIX and manually adjusted with a 617
software COOT (Emsley et al., 2010). The final Rwork was 16.6%, Rfree was 20.4%, and root 618
mean square deviations from ideal geometry of the model was 0.004 Å for bonds and 0.599 for 619
angles. The statistics for the diffraction data are listed in Table 1. The coordinates and diffraction 620
data have been deposited to the protein data bank (PDB), with 6AT7. 621
622
Enzyme kinetics 623 Kinetic parameters of wild type and mutant SbPAL1, and its isozymes were determined by 624
measuring the reaction product (cinnamate, p-coumarate, or urocanate) formation. Enzyme 625
kinetic assays were performed in a 1 mL reaction volume of 100 mM Tris buffer, pH 8.0, 626
containing 133.3 nM of the purified enzyme. Substrate (L-Phe, L-Tyr, L-His or L-DOPA) 627
concentration was varied from 0 to 10 mM. Reaction mixture without substrate was incubated at 628
30 °C and the reaction was initiated by adding the substrate to a proper final concentration. 629
Product formation was observed over 5 min at 275 nm, 310 nm, 280 nm, or 350 nm for 630
cinnamate, p-coumarate, urocanate, or caffeate, respectively. Kinetic parameters were calculated 631
with Origin 92 (OriginLab Corporation). For the inhibition kinetics, 5, and 20 µM of 2-632
aminoindan-2-phosphonic acid (AIP) was added to the reaction mixture before incubation. 633
Inhibition rate was also tested in presence of 2, 5, and 10 mM β-mercaptoethanol (βME). 634
635
Molecular docking of substrate 636
In spite of our numerous attempts to obtain complex crystals for SbPAL1 with both approaches 637
of cocrystallization and crystal soaking, we were not able to obtain any suitable complex crystal. 638
In addition, the affinity of SbPAL1 for typical inhibitor compound, 2-aminoindan-2-phosphonic 639
acid, turned out too low to be used for the complex crystallization. Therefore, to examine the 640
mode of substrate binding and the effect of His/Phe/Tyr at 123 position, in silico substrate-641
docking experiments were performed with AutoDock Vina (Trott and Olson, 2010) for L-642
phenylalanine and L-tyrosine. Prior to a docking calculation, ammonia adduct bound to the 643
methylidene carbon of MIO was removed from the SbPAL1 crystal structure and in silico 644
mutation of H123F- and H123Y-SbPAL1 was performed with COOT software. Once the 645
substrate-binding site was confirmed by a blind search where the search box contained a whole 646
wild-type SbPAL1 tetramer, both binding affinity and modes of the substrates were determined 647
by the exhaustive search within the active site pocket of wild-type, H123F- and H123Y-SbPAL1. 648
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17
For each search, an ensemble of ten docking solutions, where the predicted energy of binding 649
ranged from -6.4 to -5.7 kcal/mol, were generated. A solution of binding pose with the lowest 650
predicted energy was presented. 651
652
Isothermal Titration Calorimetry 653 Isothermal titration calorimetric reactions were carried out in a MicroCal iTC200 isothermal 654
titration calorimeter (Malvern). All titrations were performed at 25 °C and stirred at 1000 rpm. 655
The calorimetric cell contained 0.06 mM SbPAL1 in 20 mM MOPS, pH 7.7, into which an initial 656
0.8 µL ligand solution was injected, followed by 19 subsequent 2.0 µL injections of 0.6 mM 657
reaction products (cinnamate, p-coumarate, or caffeate) that were also in 20 mM MOPS, pH 7.7. 658
Both the enzyme and reaction products were titrated against the same buffer (20 mM MOPS, pH 659
7.7), prior to the ITC experiment, to account for the heats of dilution. The same calorimetric 660
method was employed for inhibitor (AIP) titration for both wild-type and H123F-, Y96F-661
SbPAL1. Origin 7 MicroCal Data Analysis software analysis package (GE Healthcare) was used 662
for ITC curve fitting. A one-set-of-sites model was employed. Curve fitting equations can be 663
found in the MicroCal iTC200 User Manual appendix. 664 665 Phylogenetic analysis 666
Phylogenetic analysis of amino acid sequences of eight SbPAL isoenzymes were conducted with 667
MEGA7 software package (Kumar et al., 2016). The evolutionary history was inferred using the 668
Minimum Evolution (ME) method (Rzhetsky and Nei, 1994). The evolutionary distances were 669
computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the 670
units of the number of amino acid substitutions per site. The ME tree was searched using the 671
Close-Neighbor-Interchange (CNI) algorithm (Nei and Kumar, 2000) at a search level of 1. The 672
Neighbor-joining algorithm (Saitou and Nei, 1987) was used to generate the initial tree. All 673
positions containing gaps and missing data were eliminated. There were a total of 689 positions 674
in the final dataset. 675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
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18
Supplemental Data 695
Supplemental Figure: Relative activity of SbPAL1 in the absence of reducing agent, and in 696
the presence of βME, TCEP, GSH. 697
698
Supplemental Figure: 699
Relative activity of SbPAL1 in the absence of reducing agent, and in the presence of βME, 700 TCEP, GSH. After incubating 133.3 nM of the purified enzyme with 10 mM of corresponding 701
reducing agent on ice for 1 hour, the activity assays were performed in a 1 mL reaction volume 702
of 100 mM Tris buffer, pH 8.0. Reaction mixture without substrate was incubated at 30 °C and 703
the reaction was initiated by adding 10 mM L-Phe. Relative catalytic activity was quantified by 704
measuring the product, cinnamate, formation over 5 min at 275 nm. Retained catalytic activity of 705
SbPAL1 in the presence of the three common reducing agents were compared relative to the 706
activity in the absence of reducing agents. All measurements were triplicated. 707
708
709
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19
Fig. 1. Ribbon diagram representing the crystal structure of SbPAL1. A) Side and top view 710 of tetrameric SbPAL1. SbPAL1 forms a homodimer in the crystallographic asymmetric unit. In 711
solution, SbPAL1 forms a homotetramer with a nearby homodimer in a two-fold symmetry. 712
Subunit A and B are shown in yellow and green, and subunits A′ and B′ are shown in red and 713
blue, respectively. B) Monomeric SbPAL1. The MIO domain is shown in cyan, the core domain 714
in beige, and the shielding domain in magenta. MIO is represented as orange ball-and-sticks. 715
Molecular graphics images were produced using the Chimera package (UCSF). 716
717
Fig. 2. The active site of SbPAL1 with docked substrates. Two substrates, L-phenylalanine 718
(blue sticks) and L-tyrosine (pink sticks) were docked in to the active site of SbPAL1. The active 719
site was completed as a tetramer in which three subunits of the tetramer participate. Subunits A, 720
B and B′ are shown in yellow, green, and blue, respectively. Molecular graphics images were 721
produced using the Chimera package (UCSF). 722
723
Fig. 3. Substrates docked into the active site of wild-type, H123F-, and H123Y-SbPAL1. A) 724
L-phenylalanine bound to SbPAL1. B) L-tyrosine bound to SbPAL1. C) L-phenylalanine 725
bound to H123F-SbPAL1. D) L-tyrosine bound to H123F-SbPAL1. E) L-phenylalanine 726 bound to H123Y-SbPAL1. F) L-tyrosine bound to H123Y-SbPAL1. Three enzyme subunits 727
that make up the active site are shown in gray, gold and coral. Interactions between the bound 728
substrate and nearby residues are marked with dotted lines. Molecular graphics images were 729
produced using the Chimera package (UCSF). 730
731
Fig. 4. SbPAL1 Kinetics. A) Lineweaver-Burk Plot of SbPAL1 in the presence of different 732 concentrations of β-mercaptoethanol (βME). Catalysis of L-phenylalanine deamination was 733
inhibited over increasing concentration of βME. Enzymatic reaction was carried out in the 734
presence of 10 mM (○), 5 mM (▲), 2 mM (■), and 0 mM (●) of βME. B) Kinetic parameters of 735
wild-type SbPAL1, its mutants and two isozymes. The kinetic parameters; kcat, KM, and 736
kcat/KM are compared for wild-type SbPAL1, five SbPAL1 mutants (H123F, H123Y, F102Y, 737
K443E, Y96F) and two SbPAL isozymes (Sb06g022740, Sb04g026520). 738
739
Fig. 5. Isothermal titration calorimetric assay. A) Binding affinity between wild-type, 740
Y96F-SbPAL1 and reaction products. Trend of heats released by serial injections of cinnamate 741
(■), caffeate (●), and p-coumarate (▲) indicate that p-coumarate (Kd =2.14 µM) binds tighter to 742
SbPAL1 than cinnamate (34.1 µM) and caffeate (6.21 µM). Y96F-SbPAL1 displayed no affinity 743
to either cinnamate (○) or p-coumarate (□). B) Binding affinity between wild-type, H123F-744
SbPAL1 and 2-aminoindan-2-phosphonic acid (AIP). PAL specific inhibitor, AIP, showed 745
higher affinity to H123F-SbPAL1 mutant (■, Kd = 0.26 µM) than the wild-type (○). Kd for the 746
wild-type was above ITC detection limit, hence the value is not stated. Solid lines represent the 747
least square fits to the data. C) Lineweaver-Burk Plot of SbPAL1 in the presence of different 748
concentrations of 2-aminoindan-2-phosphonic acid (AIP). Enzymatic activity was inhibited 749
over increasing concentration of AIP. Enzymatic reaction was carried out in the presence of 20 750
µM (▲), 5 µM (■), and 0 µM (●) of AIP. Ki calculated from kinetics experiment was 0.22 mM. 751
752
Fig. 6. Sequence alignment of structural homologs. Sequences of SbPAL1, Petroselinum 753
crispum PAL (PDB ID: 1W27), Taxus chinensis PAM (4C6G), Rhodosporidium toruloides PAL 754
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20
(1Y2M), Nostoc punctiforme PAL (2NYF), Streptomyces globisporus TAM (2RJR), Anabaena 755
variabilis PAL (2NYN), Pantoea agglomerans PAM (3UNV), Streptomyces globisporus TAM 756
(2OHY), and Pseudomonas putida HAL (1B8F) are aligned. The outer lid-loop and shielding 757
domains are indicated with red and black boxes, respectively. 758
759
Fig. 7. PAL isozymes of Sorghum bicolor. A) Sequence alignment. Eight PAL isomers are 760
aligned; Sb04g026510 is SbPAL1. The regions of alpha-helix and beta-strand are represented 761
with coil and arrow bars, respectively. Corresponding positions of SbPAL1 His123 residue was 762
highlighted with green for all eight isomers. B) Phylogenetic tree. The optimal tree with the sum 763
of branch length of 0.573 is shown. Branch length represents the evolutionary distances that were 764
computed using the Poisson correction method and are in the unit of amino acid substitutions per 765
site. The Evolutionary history was inferred using the Minimum Evolution method in MEGA7 766
(Kumar et al., 2016) that uses the Close-Neighbor-Interchange algorithm at a search level of 1. 767
768
Fig. 8. Mechanism of SbPAL1. A) L-Phenylalanine deamination. The methylidene 769
electrophile of MIO is attacked by the ortho-carbon of the substrate, L-phenylalanine, initiating a 770
Friedel-Crafts-type deamination. B) L-Tyrosine deamination. Due to the different substrate-771
binding mode, the methylidene electrophile of MIO is attacked by the amide nitrogen of the 772
substrate, initiating an E2 reaction. 773
774
775
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21
Acknowledgements: 776 We thank Tammy Gries and Manny Saluja for their technical assistance on experiments 777
presented in this paper. The US Department of Agriculture, Agricultural Research Service, is an 778
equal opportunity/affirmative action employer and all agency services are available without 779
discrimination. Mention of commercial products and organizations in this manuscript is solely to 780
provide specific information. It does not constitute endorsement by USDA-ARS over other 781
products and organizations not mentioned. We also thank Drs. John Ralph and Hoon Kim for 2-782
aminoindan-2-phosphonic acid (AIP). 783
784
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22
Table 1. X-ray diffraction data and refinement statistics for SbPAL1 (PDB: 6AT7). 785
SbPAL1
Data Collectiona
Space group I4122
Cell dimensions
a, b, c (Å) 126.304, 126.304, 337.477
α, β, γ (°) 90.00, 90.00, 90.00
Resolution (Å) 44.66– 2.49 (2.54- 2.49)
Wavelength (Å) 1.00
Asymmetric unit 2
Total reflections 639,487
Completeness (%) 99.75 (98.8)
I/σI 22.57 (3.24)
CC1/2b 0.995 (0.854)
Redundancy 13.0
Rmeasc 0.109 (0.929)
Rpim d 0.030 (0.255)
Refinement
Resolution (Å) 44.66 - 2.49 (2.56-2.49)
Unique reflections 47,930
Rwork / Rfreee 0.160 / 0.202 (0.181 / 0.276)
B-factors (Å2)
All atoms 33.6
Solvent 38.2
R.m.s deviations
Bonds (Å) 0.003
Angles (º) 0.600
Ramachandran (%)
Favored 96.95
Outliers 0.45
Number of atoms
Protein and ligand 10,185
Water 446
786 a Numbers in parentheses refer to the highest resolution shell. 787 b CC1/2 is the correlation between two data sets each based on half of the data as defined in (Karplus and 788 Diederichs, 2012). 789 c Rmeas is the multiplicity-weighted merging R factor. 790 d Rpim is the precision indicating merging R factor. 791 e Rfree was calculated as for Rcryst using 5% of the data that was excluded from refinement. 792
793
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23
Table 2. Kinetic activity of wild-type and mutant SbPAL1 and two isozymes 794
kcat (s-1
) KM (mM) kcat/KM (s-1
mM-1
)
Wild-type
Phe 1.76 ± 0.037 0.34 ± 0.030 5.18
Tyr 0.31 ± 0.004 0.12 ± 0.009 2.52
L-DOPA 0.30 ± 0.006 0.40 ±0.034 0.76
H123F Phe 1.97 ± 0.032 0.06 ± 0.006 32.29
Tyr NA* NA NA
H123Y Phe 0.17 ± 0.010 0.23 ± 0.063 0.72
Tyr NA NA NA
F102Y Phe 2.01 ± 0.092 2.57 ± 0.305 0.78
Tyr 1.11 ± 0.051 2.89 ± 0.334 0.38
K443E Phe NA NA NA
Tyr NA NA NA
Y96F Phe NA NA NA
Tyr NA NA NA
Sb06g022740 Phe 2.34 ± 0.096 4.02 ± 0.376 0.58
Tyr 0.37 ± 0.008 0.42 ± 0.037 0.89
Sb04g026520 Phe 2.20 ± 0.050 1.52 ± 0.102 1.45
Tyr NA NA NA
wild type and mutants showed no catalytic activity with histidine
* NA (no activity) indicates there was no measurable activity
795
796
797
798
799
800
801
802
803
804
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24
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Fig. 1.
A.
B.
Fig. 1. Ribbon diagram representing the crystal structure of SbPAL1. A) Side and top
view of tetrameric SbPAL1. SbPAL1 forms a homodimer in the crystallographic
asymmetric unit. In solution, SbPAL1 forms a homotetramer with a nearby homodimer in a
two-fold symmetry. Subunit A and B are shown in yellow and green, and subunits A′ and B′
are shown in red and blue, respectively. B) Monomeric SbPAL1. The MIO domain is shown
in cyan, the core domain in beige, and the shielding domain in magenta. MIO is represented
as orange ball-and-sticks. Molecular graphics images were produced using the Chimera
package (UCSF).
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Fig. 2.
Fig. 2. The active site of SbPAL1 with docked substrates. Two substrates, L-phenylalanine
(blue sticks) and L-tyrosine (pink sticks) were docked in to the active site of SbPAL1. The
active site was completed as a tetramer in which three subunits of the tetramer participate.
Subunits A, B and B′ are shown in yellow, green, and blue, respectively. Molecular graphics
images were produced using the Chimera package (UCSF).
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Fig. 3.
Fig. 3. Substrates docked into the active site of wild-type, H123F-, and H123Y-SbPAL1.
A) L-phenylalanine bound to SbPAL1. B) L-tyrosine bound to SbPAL1. C) L-
phenylalanine bound to H123F-SbPAL1. D) L-tyrosine bound to H123F-SbPAL1. E) L-
phenylalanine bound to H123Y-SbPAL1. F) L-tyrosine bound to H123Y-SbPAL1. Three
enzyme subunits that make up the active site are shown in gray, gold and coral. Interactions
between the bound substrate and nearby residues are marked with dotted lines. Molecular
graphics images were produced using the Chimera package (UCSF).
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Fig. 4.
A.
B.
Fig. 4. SbPAL1 Kinetics. A) Lineweaver-Burk Plot of SbPAL1 in the presence of different
concentrations of β-mercaptoethanol (βME). Catalysis of L-phenylalanine deamination
was inhibited over increasing concentration of βME. Enzymatic reaction was carried out in the
presence of 10 mM (○), 5 mM (▲), 2 mM (■), and 0 mM (●) of βME. B) Kinetic parameters
of wild-type SbPAL1, its mutants and two isozymes. The kinetic parameters; kcat, KM, and
kcat/KM are compared for wild-type SbPAL1, five SbPAL1 mutants (H123F, H123Y, F102Y,
K443E, Y96F) and two SbPAL isozymes (Sb06g022740, Sb04g026520).
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Fig. 5.
A. B.
C.
Fig. 5. Isothermal titration calorimetric assay. A) Binding affinity between wild-type,
Y96F-SbPAL1 and reaction products. Trend of heats released by serial injections of
cinnamate (■), caffeate (●), and p-coumarate (▲) indicate that p-coumarate (Kd =2.14 µM)
binds tighter to SbPAL1 than cinnamate (34.1 µM) and caffeate (6.21 µM). Y96F-SbPAL1
displayed no affinity to either cinnamate (○) or p-coumarate (□). B) Binding affinity between
wild-type, H123F-SbPAL1 and 2-aminoindan-2-phosphonic acid (AIP). PAL specific
inhibitor, AIP, showed higher affinity to H123F-SbPAL1 mutant (■, Kd = 0.26 µM) than the
wild-type (○). Kd for the wild-type was above ITC detection limit, hence the value is not stated.
Solid lines represent the least square fits to the data. C) Lineweaver-Burk Plot of SbPAL1 in
the presence of different concentrations of 2-aminoindan-2-phosphonic acid (AIP).
Enzymatic activity was inhibited over increasing concentration of AIP. Enzymatic reaction was
carried out in the presence of 20 µM (▲), 5 µM (■), and 0 µM (●) of AIP. Ki calculated from
kinetics experiment was 0.22 mM.
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Fig. 6.
Fig. 6. Sequence alignment of structural homologs. Sequences of SbPAL1, Petroselinum
crispum PAL (PDB ID: 1W27), Taxus chinensis PAM (4C6G), Rhodosporidium toruloides
PAL (1Y2M), Nostoc punctiforme PAL (2NYF), Streptomyces globisporus TAM (2RJR),
Anabaena variabilis PAL (2NYN), Pantoea agglomerans PAM (3UNV), Streptomyces
globisporus TAM (2OHY), and Pseudomonas putida HAL (1B8F) are aligned. The outer lid-
loop and shielding domains are indicated with red and black boxes, respectively.
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Fig. 7.
A. B.
Fig. 7. PAL isozymes of Sorghum bicolor. A) Sequence alignment. Eight PAL isomers are
aligned; Sb04g026510 is SbPAL1. The regions of alpha-helix and beta-strand are represented
with coil and arrow bars, respectively. Corresponding positions of SbPAL1 His123 residue was
highlighted with green for all eight isomers. B) Phylogenetic tree. The optimal tree with the
sum of branch length of 0.573 is shown. Branch length represents the evolutionary distances
that were computed using the Poisson correction method and are in the unit of amino acid
substitutions per site. The Evolutionary history was inferred using the Minimum Evolution
method in MEGA7 (Kumar et al., 2016) that uses the Close-Neighbor-Interchange algorithm
at a search level of 1.
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Fig. 8.
A.
B.
Fig. 8. Mechanism of SbPAL1. A) L-Phenylalanine deamination. The methylidene
electrophile of MIO is attacked by the ortho-carbon of the substrate, L-phenylalanine, initiating
a Friedel-Crafts-type deamination. B) L-Tyrosine deamination. Due to the different substrate-
binding mode, the methylidene electrophile of MIO is attacked by the amide nitrogen of the
substrate, initiating an E2 reaction.
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