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Amino-4-imidazolecarboxamide ribotide (AICAR) directly inhibits coenzyme A 2
biosynthesis in Salmonella enterica 3
4
5
Jannell V. Bazurto and Diana M. Downs* 6
7
Department of Microbiology 8
University of Georgia 9
Athens, GA 30602 10
11
12
Running title: AICAR inhibits CoA biosynthesis 13
14
Keywords: amino-4-imidazolecarboxamide ribotide (AICAR), coenzyme A (CoA) 15
biosynthesis, PanC, pantoate く-alanine ligase, pantothenate synthetase, metabolic 16
crosstalk, thiamine biosynthesis, phosphoribosylanthranilate (PR-Ant). 17
18
*Corresponding Author: 19
Department of Microbiology 20
University of Georgia 21
120 Cedar St. 22
Athens, GA 30602 23
PH: 706-542-1434 24
FAX: 706-542-2674 25
Email: [email protected] 26
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JB Accepts, published online ahead of print on 2 December 2013J. Bacteriol. doi:10.1128/JB.01087-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 29
Aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic 30
intermediate and a byproduct of histidine biosynthesis. In bacteria, yeast and humans, 31
accumulation of AICAR has been shown to affect an array of cellular processes by both 32
direct and indirect mechanisms. In purine biosynthesis, AICAR is the substrate of the 33
bifunctional protein phosphoribosylaminoimidazolecarboxamide formyltransferase /IMP 34
cyclohydrolase (PurH, E.C. 2.1.2.3/3.5.4.10). Strains lacking PurH accumulate AICAR 35
and have a defect in the synthesis of the 4-amino-5-hydroxymethyl-2-methylpyrimidine 36
(HMP) moiety of thiamine. The formation of HMP is also compromised in vivo when 37
CoA levels are reduced. Results herein showed that the in vivo accumulation of AICAR 38
decreased total CoA pools and further, AICAR inhibited the activity of pantoate く-39
alanine ligase in vitro (PanC, E.C. 6.3.2.1). These results defined a mechanism of AICAR 40
action and provided new insights into the metabolic consequences of disrupting purine 41
metabolism. 42
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INTRODUCTION: 45
Historically cellular metabolism has been described in the context of discrete 46
metabolic units (e.g., biosynthetic or degradative pathways), a perspective that facilitated 47
efforts to understand metabolic components like enzymes and metabolites. In reality 48
these units are integrated to form a complex metabolic network whose connectivity is 49
mediated extensively by metabolites. Under appropriate circumstances (i.e., growth 50
conditions or strain background), dynamic changes of intracellular metabolite levels can 51
have significant impacts on cellular fitness, by providing unexpected mechanisms for 52
survival (1, 2) or compromising growth. Dissecting metabolic integration in bacteria, and 53
understanding the diverse roles of metabolites, can reveal paradigms that are conserved 54
across phylogenetic kingdoms. Defining these paradigms provides insights into the 55
consequences of perturbing the metabolic network that can be exploited for desired 56
outcomes. 57
Amino-4-imidazolecarboxamide ribotide (AICAR) is an intermediate in the de novo 58
purine biosynthetic pathway and its accumulation impacts a variety of metabolic 59
processes in a number of organisms such as S. enterica (3-6), Escherichia coli (8), S. 60
cerevisiae (9), and humans (11). AICAR is also generated as a byproduct during histidine 61
biosynthesis and recycled by subsequent entry into the purine biosynthetic pathway in 62
organisms that synthesize histidine, (Figure 1) (12-14). Much of the work defining the 63
consequences of AICAR accumulation has been in eukaryotic cell lines, where AICAR 64
has been associated with disease treatments, such as methotrexate, and has shown 65
therapeutic potential with regard to carbohydrate and lipid metabolism (15-19) and tumor 66
cell proliferation (20, 21). In bacteria, AICAR is linked to metabolic disturbances in 67
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thiamine biosynthesis (3-5), gluconeogenesis (6) and purine metabolism (8). In cases 68
where it has been characterized, the mechanism of AICAR action is conserved between 69
humans and bacteria, a finding that emphasizes the value of understanding the diverse 70
targets of AICAR in a bacterial system. 71
Phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase 72
(PurH, E.C. 2.1.2.3/3.5.4.10) is the bifunctional enzyme that catalyzes the last two steps 73
in purine biosynthesis and uses AICAR as a substrate (Figure 1). S. enterica strains 74
lacking PurH accumulate AICAR and have a requirement for exogenous thiamine (3-5). 75
Thiamine pyrophosphate (TPP) is an essential coenzyme required for central metabolic 76
enzymes such as pyruvate dehydrogenase (E.C. 1.2.4.1), α-ketoglutarate dehydrogenase 77
(E.C. 1.2.4.2) and transketolase (E.C. 2.2.1.1). TPP is synthesized by convergent 78
pathways; each of which generates a ringed moiety; either 4-methyl-5-(beta-79
hydroxyethyl)-thiazole (THZ) monophosphate or 4-amino-5-hydroxymethyl-2-80
methylpyrimidine (HMP) pyrophosphate. The THZ and HMP moieties are then 81
condensed to form thiamine monophosphate, which is phosphorylated to the coenzymic 82
form, TPP (22). The first five steps of the HMP biosynthetic pathway are common to the 83
purine biosynthetic pathway. 84
Despite the fact that PurH functions beyond the branchpoint to HMP synthesis, purH 85
mutants require the HMP moiety of thiamine (3-5, 14). Previous studies showed the 86
requirement for HMP was caused by accumulated AICAR, and due to a constraint in the 87
conversion of amidoimidazole ribotide (AIR) to HMP (14). AIR is converted to HMP by 88
the S-adenosylmethionine radical protein ThiC (23, 24). In vivo, decreased coenzyme A 89
(CoA) levels result in lowered ThiC activity (14, 25) yet in vitro, ThiC activity does not 90
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require, and is not improved by, the addition of CoA to the reaction (26). 91
CoA is a universally required coenzyme and an important carrier molecule that has a 92
pivotal role in metabolic processes such as fatty acid oxidation and synthesis, the 93
tricarboxylic acid cycle and amino acid biosynthesis. CoA is composed of adenosine 94
monophosphate and 4’-phosphopantetheine moieties. The 4’-phosphopantetheine moiety 95
is synthesized from pathways that converge at pantoate く-alanine ligase (PanC, E.C. 96
6.3.2.1), which generates pantothenate (Figure 1). Subsequent reactions convert 97
pantothenate to 4’-phosphopantetheine, which is then adenylated by adenosine 98
triphosphate and lastly phosphorylated to generate CoA. 99
Herein we show that accumulation of AICAR decreased the cellular pool size of 100
CoA in S. enterica, and identified pantoate β-alanine ligase as the target of this effect. 101
The data showed that the thiamine requirement of a purH mutant was at least partially 102
modulated by the resulting decrease in CoA levels. This work showed that a consequence 103
of accumulating the metabolite AICAR is the disruption of CoA biosynthesis, thus 104
impacting a central component in all metabolisms. 105
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MATERIALS AND METHODS: 108
Bacterial strains, media and chemicals. 109
Strains used in this study are derivatives of S. enterica serovar Typhimurium LT2 and 110
are described in Table 1. Rich (NB) medium was made with Difco nutrient broth (8 g/L) 111
and NaCl (5 g/L). Super broth (SB) was used for protein overexpression. Defined 112
medium was no carbon E medium (NCE) supplemented with 1 mM MgSO4 (27-29) trace 113
minerals (30) and 11 mM glucose (or 20 mM ribose) as a sole carbon source. 114
Alternatively, the defined medium contained 1 mM glutamine (instead of NaNH4PO4) as 115
a limiting ammonium nitrogen source (31, 32). Difco BiTek agar (15 g/L) was added for 116
solid medium. When present in the media, compounds were at the following final 117
concentrations: adenine (0.4 mM); pantothenate (0.1 mM); histidine (0.1 mM); thiamine 118
(100 nM); ketoisovalerate (0.5 mM); ketopantoate (0.1 mM); pantoate (0.1 mM); く-119
alanine (0.1 mM); aspartate (0.3 mM). Antiobiotics used were at the following 120
concentrations for rich (minimal) medium, respectively: tetracycline, 20 (10) µg/mL; 121
kanamycin 50 (150) µg/mL; chloramphenicol, 20 (5) µg/mL; ampicillin, 100 (15) µg/mL. 122
Growth quantitation. 123
Cell cultures grown overnight in NB medium were pelleted and resuspended in an 124
equal volume of saline (0.85% NaCl). A 0.1 mL aliquot was used to inoculate 5 mL of 125
the appropriate minimal media. When the growth was quantified in a microplate reader 126
(model EL808, Bio-tek Instruments), a 5 µL inoculum was used for 195 µL of the 127
appropriate minimal media. Unless otherwise stated, cell density was measured as 128
absorbance at 650 nm and growth was routinely reported as specific growth rates (µ = 129
ln(X/X0)/T) or the final cell density (OD650) reached after 12-24 hr incubation at 37oC 130
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with shaking (33). When nutritional requirements were measured on solid medium, soft 131
agar overlays were used as has been described (14). 132
Genetic techniques. 133
General. Transductional crosses were done with a high-frequency general transducing 134
mutant of bacteriophage P22 (HT105/1, int-201) (34). Methods for transductional 135
crosses, isolation of transductants from phage and identification of phage-free 136
transductants have been previously described (35, 36). Mutant strains were constructed 137
using standard genetic techniques. 138
Isolation of suppressor mutations. Multiple cultures of DM2 (purH355) were grown 139
overnight in NB medium. Cells were pelleted and resuspended in an equal volume of 140
saline. A 0.1 mL aliquot (~ 1x108 cells) was spread on solid glucose medium with 141
adenine (28). After incubation at 37oC for 48-72 hours, colonies that arose were streaked 142
for isolation on non-selective rich medium and were further characterized. To ensure 143
genetic independence, only one colony from each plate was pursued. 144
Cloning panC. 145
The panC gene was amplified from wild-type S. enterica (DM10000). The primers 146
were flanked by EcoRI (forward) and XhoI (reverse) restriction sequences. The blunt-147
ended amplicon was cloned with the StrataClone Blunt PCR Cloning Kit (Agilent 148
Technologies). The vectors from transformants were isolated and screened for the panC 149
insertion. The panC insertion was excised with EcoRI and XhoI and subcloned into pET-150
20b(+) digested with the same restriction enzymes. 151
Purification of PanC. 152
A 50 mL overnight culture (at room temperature with shaking) of BL21-AI 153
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(transformed with pET20b-panC) in SB supplemented with 150 µg/mL ampicillin was 154
used to inoculate two flasks (each with 1.5 L) of the same medium. Arabinose was added 155
to 0.2 % when the OD650 reached 0.5 and incubation continued for 12 hours at room 156
temperature prior to harvesting the cells by centrifugation (7000 g for 10 min. at 4 °C). 157
The cell pellet was resuspended in binding buffer (50 mM KPO4, pH 7.5, 100 mM KCl, 158
5% glycerol). DNAse and lysozyme were added at 0.01 mg/mL, cells were broken with a 159
French pressure cell press (three passes at 1500 psi), and cell debris was pelleted by 160
centrifugation (18,000 g for 45 min at 4 ºC). Lysate was passed through a 0.22 µm filter 161
and loaded onto Ni-NTA Superflow resin (Qiagen). Five column volumes (CV) of 162
binding buffer, followed by 15 CV of binding buffer containing 15 mM imidazole were 163
passed over the column until the absorbance (280 nm) of the eluent was zero. PanC was 164
eluted with ~ 100 mM imidazole when a gradient (15-500 mM imidazole) was applied 165
over 20 CV (200 mL). PanC concentration was determined using the bicinchoninic acid 166
assay (Pierce). TotalLab Quant software was used to determine that the protein purity 167
was > 99%. 168
PanC activity assays. 169
General. PanC activity was assayed by modifying a previously described method where 170
AMP formation is coupled to myokinase and lactic dehydrogenase activities (37, 38). 171
Ultimately, two molecules of NADH were oxidized per molecule of pantothenate formed 172
Final reaction volumes were 200 µL and contained the following: 100 mM Tris, pH 7.5, 173
10 mM MgCl2, 0.3 mM NADH, 2.5 mM ATP, 1.5 mM phophoenolpyruvate, 3 mM ß-174
alanine, 4 mM pantoate, 50 u/mL pyruvate kinase, 55 u/mL lactate dehydrogenase, 25 175
u/mL myokinase, and 50 nM PanC. The oxidation of NADH was monitored at 340 nm in 176
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a microplate reader (SpectraMax Plus, Molecular Devices). The linear equation of an 177
NADH standard curve was generated and used to convert absorbance to the appropriate 178
units. The data were fit to the Michaelis-Menten model in GraphPad Prism version 6.0b 179
(www.graphpad.com). 180
Inhibition studies. A screen of potential inhibitors of PanC (AICAR, AICARs, or AIC) 181
was performed by their inclusion in the reaction mixtures at 3 and 5 mM. Reactions were 182
initiated by the addition of pantoate (40 µL). Inhibition of PanC by AICAR was further 183
characterized using five AICAR concentrations (0-16 mM) at various concentrations of 184
pantoate (0-16 mM), ß-alanine (0-8 mM), and ATP (0-4 mM). Reactions to address 185
inhibition by AICAR were initiated by the addition of the substrate that was being varied 186
and ATP (52-85 µL). Lineweaver-Burk plots were used to determine the form of 187
inhibition for each substrate. Substrate-velocity data were then fit to the corresponding 188
inhibition models (GraphPad Prism version 6.0b) to determine Ki values. Each fit was 189
constrained by experimentally derived Km values for the relevant substrate: pantoate 190
(1.39 mM), ß-alanine (0.66 mM), ATP (1.62 mM). 191
Total coenzyme A measurements. 192
Biological triplicates of relevant strains were grown to an OD650 of approximately 0.5 193
in 200 mL of minimal glucose (16.5 mM) medium containing adenine and thiamine with 194
or without 0.1 mM hisitidine. Total cellular CoA pools were quantitated by a previously 195
described method (39). NADH formation was monitored at 340 nm in a traditional 196
UV/Vis spectrometer (Lambda Bio 40, Perkin Elmer Instruments) or in a microplate 197
reader (SpectraMax Plus, Molecular Devices). 198
ȕ-galactosidase assays. 199
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Biological triplicates of DM308 (panC607::MudJ), DM309 (panC607::MudJ 200
panB614::Tn10d(Tc), and purH isogenic strains DM14375 (purH355 panC607::MudJ) 201
and DM14376 (panC607::MudJ) were grown to OD650 between 0.4 - 0.5 in 5 mL of 202
minimal glucose medium containing adenine, pantothenate, and thiamine with or without 203
0.1 mM hisitidine. く-galactosidase activity of each sample was quantitated by a 204
previously described method (40-42) 2-nitrophenol formation was monitored at 420 nm 205
in a microplate reader (SpectraMax Plus, Molecular Devices) for 60 min. The rates of 206
product formation were determined as Abs420/(min OD650). 207
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RESULTS : 210
AICAIR inhibits multiple pathways important for HMP synthesis. DM2 (purH355) 211
is a representative purH mutant strain with a previously described frameshift mutation 212
that eliminates all PurH activity in the cell (6). DM2 and other purH mutants of 213
S.enterica required exogenous addition of the HMP moiety of thiamine to grow in 214
glucose medium with adenine (3-5). Data in Figure 2 show the nutritional additions that 215
partially or fully eliminate the need for exogenous thiamine. These data confirmed 216
previous observations that histidine, methionine and pantothenate each individually 217
restored growth (5, 12) (43), and showed that methionine and pantothenate were additive 218
in their effect. Because exogenous histidine lowers AICAR in the cell (5, 12, 14), these 219
data emphasized that AICAR accumulation was the metabolic imbalance that caused a 220
thiamine requirement and that more than one metabolic pathway was disrupted by this 221
accumulation. The stimulatory effect of exogenous pantothenate suggested an 222
involvement of CoA levels in the growth phenotype (Figure 1). 223
CoA levels are decreased in a purH mutant. The reported link between CoA levels and 224
ThiC activity (14) in combination with the results above suggested that the accumulation 225
of AICAR negatively impacted CoA biosynthesis. Total CoA was quantified in wild-type 226
and purH mutant strains and the data are shown in Table 2. Significantly, in glucose 227
adenine medium where the strain requires exogenous thiamine, the purH mutant had total 228
CoA levels three-fold lower than the wild-type strain. Furthermore, addition of 229
exogenous histidine restored wild-type levels of CoA by reducing the formation of 230
AICAR, which eliminated the requirement for exogenous thiamine. These results 231
established an inverse correlation between AICAR accumulation and CoA levels in the 232
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cell. With the knowledge that decreased CoA levels cause reduced ThiC activity, these 233
data supported the hypothesis that AICAR inhibits HMP synthesis by lowering cellular 234
CoA pools. 235
Pantothenate biosynthesis is compromised in a purH mutant. The stimulation of 236
thiamine-independent growth in a purH mutant by pantothenate was consistent with a 237
constraint in CoA biosynthesis that was prior to the formation of pantothenate. 238
Pantothenate is formed by the condensation of two separately synthesized moieties, 239
pantoate and く-alanine. Each moiety was provided as a supplement, and thiamine-240
independent growth of DM2 was quantified in liquid media. The data in Figure 3 showed 241
that when both pantoate and く-alanine were present, growth was restored to the level 242
allowed by the addition of pantothenate. The requirement for both pantoate and く-alanine 243
suggested that either AICAR inhibited biosynthesis of both moieties or, that the PanC 244
reaction was compromised. Further nutritional studies showed that providing both 245
ketoisovalerate and aspartate to minimal glucose adenine medium allowed thiamine-246
independent growth of DM2 (た = 0.28), suggesting that the biosynthetic enzymes 247
upstream of PanC were not compromised. 248
When panC was provided in trans (pET20b-panC) under control of a constitutive 249
promoter thiamine-independent growth of the purH mutant was not restored (data not 250
shown). Importantly, pET20b-panC was able to complement a panC355 mutant (DM3) 251
in minimal glucose adenine medium. The expression of the panBCD operon was 252
quantitated by く-galactosidase assays with strains containing panC607::MudJ in the 253
chromosome. The rate of く-galactosidase product formation in DM14375 (purH355, 254
0.846 Abs420/(min OD650)) was comparable to the isogenic strain DM14376 (0.688 255
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Abs420/(min OD650)). These results eliminated scenarios in which AICAR decreased gene 256
expression of the pan operon or the panC gene specifically. 257
AICAR inhibits the PanC reaction. A number of enzymes are inhibited allosterically 258
by AICAR, including several that bind adenosine (11) or ATP (6). Based on this 259
precedent and the involvement of ATP in the PanC reaction mechanism, it was plausible 260
that PanC was directly inhibited by AICAR. PanC was purified and assayed in vitro with 261
and without AICAR and its derivatives. AICAR inhibited the PanC reaction when 262
provided in concentrations similar to that of the substrates, reducing activity by ~3-fold 263
(Table 3). The AICAR riboside (AICARs) reduced PanC activity by ~ 1.5-fold, while 264
AIC, the free base of AICAR, did not have any significant effect on the reaction. 265
Inhibition by AICAR was probed further and found to be noncompetitive with pantoate 266
(Ki=5.7 mM), uncompetitive with く-alanine (Ki’=4.1 mM), and competitive with respect 267
to ATP (Ki=1.8 mM). These data are shown in Figure 4 and were consistent with 268
previous reports of the kinetic mechanism of E. coli PanC (38, 44). These in vitro data 269
supported the hypothesis that when AICAR (and/or its riboside) accumulate, CoA levels 270
are reduced in vivo due to a direct inhibition of the PanC-catalyzed reaction. 271
Suppressor analyses implicate CoA levels as source of HMP requirement. In vivo 272
suppressor analyses were initiated to dissect the role of CoA in the AIR to HMP 273
conversion. Specifically, we sought to identify mutations that allowed thiamine synthesis 274
despite low CoA levels. Forty independent spontaneous mutations that allowed a purH to 275
grow on glucose adenine medium were characterized and assigned to one of three classes 276
(Figure 5A). 277
Mutations in panR (the promoter region of the 3 min. panBCD operon) that restored 278
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thiamine-independent growth to a purH mutant have been previously described (45). To 279
identify panR mutations among the new suppressor strains, the strains were screened for 280
an ability to feed a pantothenate auxotroph (DM3547; panC::Tn10d(Tc)). Of the forty 281
isolates, 29 (>70%) contained a panR mutation based on this assay. A representative 282
seven of these strains were tested to confirm the location of the mutations. In each case 283
the causative mutation was co-transducible with a characterized insertion 284
(zae3653::Tn10) near the panBCD locus. Sequence analysis determined six of the seven 285
strains carried the same base addition that was previously shown to increase transcription 286
of the pan operon (46). The seventh strain carried a transition (C to T) mutation two base 287
pairs upstream of the transcription start site for panBCD (46). While not further 288
characterized, this mutation brought the promoter sequence closer to consensus and was 289
thus expected to increase transcription of the pan operon. This class of mutations allowed 290
thiamine synthesis by restoring CoA levels. 291
The remaining eleven revertant strains were screened for the ability to utilize 292
succinate. Loss of function mutations in sdh (encoding succinate dehydrogenase) restore 293
thiamine-independent growth to a variety of strains with compromised thiamine 294
biosynthesis by lowering the cellular thiamine requirement (47). Eight of the eleven 295
strains were unable to utilize succinate, leaving three purH revertants (DM8384, 296
DM8388, DM8397) that were not in a previously described class. 297
Increased AIR formation bypasses reduced CoA in HMP synthesis. Genetic analysis 298
of the remaining three mutations placed them in the trp operon and sequence analysis 299
identified each of the mutations in trpC (Table 4). The relevant trpC alleles were 300
recessive, the protein variants retained sufficient activity to allow tryptophan synthesis 301
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for growth, and a role for biosynthetic flux was indicated by the negative effect of 302
tryptophan addition (Figure 5B). The data above were reminiscent of similar trpC alleles 303
that restored the synthesis of the thiamine intermediate phosphoribosylamine (PRA) by 304
the accumulation and subsequent breakdown of phosphoribosylanthranilate (PR-Ant) 305
(48). The PR-Ant breakdown product, ribose-5-phosphate, and ammonium non-306
enzymatically combine to form PRA (Figure 6). Non-enzymatic PRA formation is 307
elevated when ribose is the sole carbon source and decreased in limiting ammonium 308
medium (32, 48, 49). 309
Nutritional studies showed that while minimal NCE glucose adenine medium allowed 310
thiamine-independent growth of the purH trpC mutant (た = 0.27), limiting ammonium 311
glucose medium did not (た < 0.02). Importantly, the addition of thiamine restored growth 312
of the purH trpC mutant in limiting ammonium medium (た = 0.25). In addition, the purH 313
mutant gained the ability to grow independent of thiamine in minimal NCE medium 314
containing adenine when ribose was the sole carbon source (た = 0.23). Taken together, 315
these data suggested that increased PRA/AIR formation (via the trpC alleles or ribose) 316
suppressed the thiamine requirement of a purH mutant and allowed HMP synthesis in the 317
presence of reduced CoA levels. The desired class of mutants that impacted the need for 318
CoA in the conversion of AIR to HMP was not identified in this study. 319
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DISCUSSION: 322
323
AICAR accumulates in a purH mutant and inhibits the conversion of AIR to HMP, a 324
step catalyzed by the SAM radical protein ThiC (14). The ThiC reaction has been 325
reconstituted in vitro and is not affected by AICAR, indicating the effect in vivo was 326
indirect (26). This study was initiated to understand the mechanism by which AICAR 327
accumulation inhibited the conversion of AIR to HMP. 328
The ThiC reaction uses SAM as a co-substrate and is sensitive to fluctuations in CoA 329
levels in vivo (14, 23, 24, 50). Nutritional studies led to the hypothesis that one metabolic 330
consequence of AICAR accumulation was decreased CoA levels. Metabolite pool 331
measurements confirmed that strains that accumulated AICAR had ~3-fold lower total 332
CoA levels than the wild-type. Although this level of CoA reduction does not generate a 333
thiamine requirement in an otherwise wild-type strain, conditions that reduce flux 334
through the purine biosynthetic pathway (i.e., adenine in the medium) increase the CoA 335
requirement for thiamine synthesis (25). 336
Further nutritional studies suggested that the step in pantothenate synthesis catalyzed 337
by PanC was compromised in a purH mutant. In vitro, both AICAR and its riboside 338
inhibited PanC activity. The Ki of 1.8 mM (AICAR) measured for competitive inhibition 339
with respect to ATP is consistent with this effect being physiologically relevant. Previous 340
studies with E. coli used the antifolate trimethoprim to inhibit the AICAR transformylase 341
activity of PurH and measure the accumulation of AICAR metabolites. These data 342
showed that in 60 min. intracellular AICAR levels rose to 0.8 mM and the AICAR 343
triphosphate rose to 0.5 mM (51). Earlier work with numerous S. enterica purH mutants 344
noted that AICARs was the primary AICAR-related metabolite to accumulate (43). These 345
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data together suggested that AICAR and AICARs could inhibit PanC in vivo, which 346
would reduce pantothenate and consequently CoA production in a purH mutant. 347
Suppressor mutation analysis of the purH mutant emphasized the correlation between 348
reduced CoA and a requirement for thiamine, since deregulating pantothenate 349
biosynthesis restored thiamine-independent growth. Further, decreasing AICAR synthesis 350
with exogenous histidine restored both CoA levels and thiamine synthesis. Other 351
suppressor mutations were consistent with previous work that characterized robustness in 352
the formation of PRA, and described the thiamine sparing effect of a strain lacking 353
succinate dehydrogenase. A desired class of mutations that could provide insights into 354
the mechanism that is used by CoA to impact the conversion of AIR to HMP was not 355
found in this study and will require refined genetic screens. 356
Dissecting the biochemical mechanism(s) responsible for diverse phenotypes in 357
bacteria has repeatedly unveiled paradigms of metabolism that are conserved through a 358
multitude of organisms. The current study followed a phenotype based in thiamine 359
synthesis and led to the demonstration that CoA levels can be modulated by AICAR (and 360
its riboside), a metabolite that accumulates after various cellular perturbations in a 361
number of organisms. Results from this study have provided new insights into how CoA 362
biosynthesis is integrated into the metabolic network. Beyond the impact on CoA 363
biosynthesis, results suggest that the thiamine requirement of a purH mutant can be used 364
to define perturbation in other metabolic processes that result from the accumulation of 365
AICAR in Salmonella. Studies such as those herein will continue to define the metabolic 366
network in bacteria, and contribute to the understanding of metabolic paradigms that will 367
facilitate efforts to manipulate metabolism in a diverse organisms. 368
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ACKNOWLEDGEMENTS: 369
The authors would like to thank Mary E. Anderson for work with trpC alleles and 370
experiments probing the ability of PR-Ant breakdown products to satisfy the thiamine 371
requriement of a purH mutant. Several of the purH suppressor mutations were isolated by 372
Michael Dougherty. This work was supported by competitive grant GM47296 from the 373
NIH. Jannell Bazurto was supported by an Advanced Opportunity Fellowship and a 374
Biotechnology Traineeship from the N.I.H. (T32 GM08349). 375
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REFERENCES: 393
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thiamine synthesis in Salmonella enterica serovar Typhimurium. J. Bacteriol. 469
182:236-240. 470
26. Palmer LD, Downs DM. 2013. The thiamine biosynthetic enzyme ThiC 471
catalyzes multiple turnovers and is inhibited by S-adenosylmethionine (AdoMet) 472
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27. Vogel HJ, Bonner DM. 1956. Acetylornithinase of Escherichia coli: partial 474
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pantothenate synthetase from Escherichia coli B. J. Nutr. Sci. Vitaminol. (Tokyo) 519
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542
543
544
545
546
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547
548
549
Table 1. Bacterial strainsa
Strain Genotypeb
DM1 Wild-type
DM2 purH355
DM3 panC355
DM95 thi-885::MudJ
DM308 panC607::MudJ panB614::Tn10d(Tc)
DM309 panC607::MudJ
DM1155 panR561
DM1231 purF2085 gnd174::MudJ
DM3547 panC617::Tn10d(Tc)
DM6123 purG2324::MudJ zxx9126::Tn10d(Tc) stm4068-1 purH355
DM6124 purG2324::MudJ zxx9126::Tn10d(Tc) stm4068-1 purH355
DM8384 purH trpC3639
DM8388 purH trpC3638
DM8397 purH trpC3640
DM10338 purF2085 gnd181 trpE8
DM13784 purH355 STM1719::Tn10d(Tc) trpC3638
DM13785 purH355 STM1719::Tn10d(Tc)
DM13881 purH355 STM1719::Tn10d(Tc) trpC3639
DM13882 purH355 STM1719::Tn10d(Tc)
DM13883 purH355 STM1719::Tn10d(Tc) trpC3640
DM13884 purH355 STM1719::Tn10d(Tc)
DM14013 ǻpurH3121 purR2319::Tn10d(Tc)
DM14043 purH355 zae-3653::Tn10d(Tc) panR801
DM14044 purH355 zae-3653::Tn10d(Tc)
DM14045 purH355 zae-3653::Tn10d(Tc) panR802
DM14046 purH355 zae-3653::Tn10d(Tc)
DM14124 purH355 trpR3614::cat trpC::Tn10d(Tc) trpE3613
DM14125 purH355 trpR3614::cat trpC::Tn10d(Tc)
DM14375 panC607::MudJ zxx-8039:: Tn10d(Tc) purH355
DM14376 panC607::MudJ zxx-8039:: Tn10d(Tc) a All strains are S. enterica serovar Typhimurium LT2 and were part of the lab
collection or were constructed for this study. b Tn10d(Tc) refers to the transposition-defective mini-Tn10 (Tn10∆16∆17)
construct(7). MudJ refers to the MudJ1734 transposon (10).
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550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
Table 2. Total cellular CoA levels
Strains Media additionsa nmol CoA / DCW (avg ± SD)
Wild-type None 0.86 ± 0.05
His 1.04 ± 0.11
purH355 None 0.29 ± 0.05
His 1.04 ± 0.24
CoA levels are expressed in nmol of CoA per mg of dry cell weight.
Strains were grown in minimal medium containing glucose, adenine and
thiamine. Data shown are the average total CoA from three independent
cultures, measured in mid-log phase. a Abbreviations: His, histidine; DCW, dry cell weight.
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567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
Table 3. AICAR inhibits PanC activity
NADH oxidation (µmol/min/mg)
Putative inhibitors - inhibitor + inhibitor
AICAR 4.73 ± 0.08×10-3
1.46 ± 0.06×10-3
AICARs 5.51 ± 0.58×10-3
3.23 ± 0.10×10-3
AIC 4.28 ± 0.34×10-3
3.60 ± 0.24×10-3
PanC activity (AMP formation) was coupled to NADH
oxidation, which is expressed in µmol/min/mg. Data
represent reactions performed in triplicate. The trends were
confirmed by two independent experiments.
Abbreviations: AIC, 5-amino-4-imidazolecarboxamide;
AICAR, AIC ribotide; AICARS, AIC riboside.
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597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
Table 4. Mutations that affect PR-Ant isomerase domain of TrpC
Strain Allele Mutationa Protein Variant
a
DM8388 trpC3638 Transition (GA) A384D
DM8384 trpC3639 3 Transitions (GA) A315T, S361N, G407S
DM8397 trpC3640 Transition (GA) A403T
N-terminal residues compose the indoleglycerol phosphate synthase domain and
C-terminal residues, which include residues 315 to 407, compose the PR-Ant
isomerase domain. aNumbering began with the first amino acid of the TrpC protein.
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FIGURE LEGENDS 614
Figure 1. Connections between the purine, thiamine, histidine, and CoA biosynthetic 615
pathways in S. enterica. Biosynthetic pathways are shown schematically and relevant 616
gene products are with the reactions they catalyze. The AICAR-mediated inhibition of 617
the ThiC reaction is indirect and mediated by AICAR’s inhibition of PanC and resulting 618
decrease in CoA levels. Abbreviations: PRPP, phosphoribosyl pyrophosphate; Gln, 619
glutamine; PRA, 5-phosphoribosylamine; AIR, 5-aminoimidazole ribotide; HMP, 4-620
amino-5-hydroxymethyl-2-methylpyrimidine; Met, methionine; DOA, 5’-621
deoxyadenosine; TPP, thiamine pyrophosphate; THZ-P, 4-methyl-5-(2-622
phosphonooxyethyl)thiazole; AICAR, 5-amino-4-imidazolecarboxamide ribotide; IMP, 623
inosine monophosphate; GMP, guanosine monophosphate; AMP, adenosine 624
monophosphate; ATP, adenosine triphosphate; His, histidine; PPi, pyrophosphate. 625
626
Figure 2. Pantothenate and methionine additively restore thiamine-independent 627
growth of a purH mutant. Growth of a purH mutant was quantified in minimal medium 628
containing glucose, adenine with no addition (solid circles); methionine (triangles); 629
pantothenate (inverted triangles); methionine and pantothenate (diamonds); histidine 630
(squares); thiamine (open circles). 631
632
Figure 3. Pantoate and ȕ-alanine restore endogenous HMP biosynthesis in a purH 633
mutant. Growth of a purH mutant was quantified in minimal medium containing 634
glucose, adenine with pantoate (filled triangles); く-alanine (circles); pantoate and く-635
alanine (squares); pantothenate (triangles). 636
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Figure 4. AICAR Inhibits the PanC reaction. The PanC activity was observed with 637
AICAR present in the reactions at 0 (open circles), 2 (half-filled circles), 4 (filled 638
circles), 8 (half-filled squares), and 16 (filled squares) mM. For each concentration of 639
AICAR the concentration of one substrate was varied. The resulting substrate-velocity 640
data are shown with curves fitted to the appropriate inhibition model. A) versus ATP, 641
competitive inhibition, B) versus pantoate, noncompetitive inhibition, C) versus く-642
alanine, uncompetitive inhibition. Rate of pantothenate formation is expressed in nM/s. 643
GraphPad Prism version 6.0b was used to fit each data set to Michaelis-Menten equations 644
for inhibition and determine Ki values. 645
646
Figure 5. Suppressor mutations restored thiamine-independent growth in a purH 647
mutant. Growth of purH mutant derivatives was quantified in minimal medium 648
containing glucose, adenine. A: Representative panR (circles), sdh (squares) and trpC 649
(diamonds) derivatives were grown with thiamine (open symbols) and without thiamine 650
(closed symbols). B: Growth of the reconstructed purH trpC strain (diamonds) is shown 651
with no addition (closed symbols), thiamine (open symbols), and tryptophan (closed gray 652
symbols). The isogenic purH strain (circles) is also shown. 653
654
Figure 6. The breakdown of PR-Ant leads non-enzymatic PRA formation. 655
Biosynthetic pathways are shown schematically and relevant gene products are with the 656
reactions they catalyze. The dashed arrow indicates the PR-Ant isomerase activity of 657
TrpC which when compromised leads to the accumulation of the substrate PR-Ant. The 658
R5P breakdown product can non-enzymatically combine with ammonium to form the 659
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thiamine/purine intermediate PRA. Abbreviations: Gln, glutamine; PR-Ant, 660
phosphoribosylanthranilate; R5P, ribose-5-phosphate; PRA, 5-phosphoribosylamine; 661
AIR, 5-aminoimidazole ribotide; TPP, thiamine pyrophosphate. 662
663
664
665
666
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NN
H2N
R5PN
N
H2N
R5P
H2N
O
PRPP + Gln
PRA
PurH
His
AIR AICAR
HMP-P
TPP
THZ-P
SAM
5’DOA ThiC
N
N
H2N
2-O3PO
+ Met
Pantoate
-alanine
CoA
AMP
+
PPi
IMP
ATP
PRPP HisG
PurF
ATP
+
PurH
O
OH
OH
HO
H2N
O
OH
HO
O
OH
HN
O
OH
Pantothenate
?
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0 4 8 12 16
0.1
1
Time (hr)
Absorb
ance (650 n
m)
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0 4 8 12 16
0.1
1
Time (hr)
Absorb
ance (650 n
m)
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0 2 4 6 80
10
20
30
40
-alanine (mM)
Ra
te (n
M/s
)
0.0 0.5 1.0 1.5 2.0
0.05
0.10
0.15
0.0
1 / -alanine (mM-1)
1 /
Ra
te (s/ n
M)
-1.0 -0.5 0.0 0.5 1.0
0.05
0.15
0.25
0.0
1 / Pantoate (mM-1)
1 /
Ra
te (s/ n
M)
0 1 2 3 40
10
20
30
40
ATP (mM)
Ra
te (n
M/s
)
0 1 2 3 4
0.0
0.4
0.8
1.2
1 / ATP (mM-1)
0 4 8 12 160
10
20
30
40
Pantoate (mM)
1 /
Ra
te (s/ n
M)
Ra
te (n
M/s
)
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A B
0 4 8 12 16 20
0.1
1
Time (hr)
Absorb
ance (650 n
m)
0 4 8 12 16
0.1
1
Time (hr)
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Chorismate +
Gln TrpED N
H
O
HO
OH
2-OPO3
O-O
Tryptophan
PR-Ant
TrpC TrpC
R5P PRA
anthranilate
OH
O
HO
OH
2-OPO3
NH2
O
HO
OH
2-OPO3
TPP
NH4+
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