Supporting Information

Computer-Assisted Engineering the Catalytic

Activity of a Carboxylic Acid Reductase

Ge Qu,1,# Beibei Liu,1,# Kun Zhang,1 Yingying Jiang,1 Jinggong Guo,2 Ran Wang,3 Yuchen Miao,2 Chao Zhai,4 Zhoutong Sun1,*

1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China.2 State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng 475001, China.3 Zhengzhou Tabacco Research Institute of CNTC, No. 2 Fengyang Street, Zhengzhou 450001, Henan, China.4 State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, 368 Youyi Road, Wuchang Wuhan, 430062, China.# Authors contribute equally to this work.

* Correspondence: (+86) 22-84861981; e-mail: sunzht@tib.cas.cn.

ContentsSupporting tables.....................................................................................................S3

Table S1. Key residues lining at the two substrate binding pockets........................................S3Table S2. Screening results of single site saturation mutagenesis in each SrCAR library toward the transformation of 1 to 3. The WT enzyme (14% conversion) is used as control...S4Table S3. Catalytic activity of the mutant K524R and K524H toward the transformation of 1 to 3..........................................................................................................................................S5Table S4. List of primers for saturation mutagenesis of single residue using NDT/VMA/ATG/TGG as degenerate codons.a..........................................................................S6

Supporting figures....................................................................................................S8Figure S1. The conversion of SrCAR mutants K524W and K524W/A937V in the reaction of benzoic acid in 0.5 h and 1 h reaction time. Error bars depict standard deviations over the 3 independent replicates. The reaction was analyzed by HPLC using an Agilent ZORBAX SB C18 column (4.6 mm × 250 mm × 5 μm) at a flow rate of 1 mL/min (the ratio of acetonitrile to 0.1% TFA was 3:7) and detection at 258 nm...........................................................................S8Figure S2. Consensus analysis of SrCAR (PDB ID 5MST) with other crystallized ANL family members. The residues T265, G267, Y431, G432, T434 and D507 are indicated by red

S1

arrows. The figure was prepared by ESPript 3.0 (Robert and Gouet, 2014), using PDB database and E-value was set as 1e-6......................................................................................S9Figure S3. GC profiles of WT and the two mutants. Compounds and corresponding peaks are indicated by black arrows......................................................................................................S10Figure S4. Michaelis-Menton curve fit of the wildtype of SrCAR (A) and the mutant K524Q (B), K524W (C), K524A (D), K524C (E), K524L (F), K524M (G) and K524V (H) in the reduction of benzoic acid......................................................................................................................S11Figure S5. Comparison of biotransformation 1 with formation of 3 employing WT, K524Q and K524W with 20 mM substrate loadings.........................................................................S12Figure S6. The position 524 (colored in magenta) is located at the surface of SrCAR, observed both in the adenylation (A) and thiolation (B) conformations..............................................S13Figure S7. Traces of selected distances (d1 and d2) monitored in the MD runs of the systems regarding to WT (A), K524Q (B) and K524W (C). AMP is colored in blue, while the side chain of residue 524 located at the A domain is displayed in black...............................................S14Figure S8. Determination of dissociation constant (Kd) of AMP (A) and PP i (B) for wild-type and variant K524W by ITC. The ITC experiments were performed using a MicroCal ITC titration calorimeter (ITC200, USA). Samples were prepared in titration buffer containing 20 mM PBS (pH 7.4) and 0.1 M NaCl. The reaction cell contained a degassed solution of 25 μM purified enzyme mixed with 13 x 2 μL aliquots of 750 μM AMP and PPi, respectively.........S15Figure S9. Root mean square deviations (RMSD) measured during 300 ns MD simulations for models WT (A), K524Q (B) and K524W (C)............................................................................S16

References.................................................................................................................S17

S2

Supporting tables

Table S1. Key residues lining at the two substrate binding pockets.Residu

esProposed functionsa

T265 ATP binding siteS266 ATP binding siteG267 ATP binding siteS408 /G430 ATP binding siteY431 ATP binding siteG432 ATP binding siteT434 ATP binding siteT505 /D507 ATP binding siteY519 ATP binding siteR522 ATP binding siteK524 /V936 /A937 /M999 /Q1015 /

a The proposed roles are assigned by consensus analysis derived from previous studies (Gulick 2009; Stolterfoht et al. 2018; Stolterfoht et al. 2017)

S3

Table S2. Screening results of single site saturation mutagenesis in each SrCAR library toward the transformation of 1 to 3. The WT enzyme (14% conversion) is used as control.

Library Mutations Conv. (%)WT - 14

S266 T 33S408 I 51

G430

K 56M 34R 34L 31T 24N 23E 19S 16

T505L 80A 28

Y519

Q 60V 49P 37A 26

R522H 62K 58Y 51

K524

Q >99W >99M 90L 51C 48A 30V 23

V936

Y 51F 31Q 30W 19

A937V 85S 84F 67

Q1015 W 33F 27G 24K 20

S4

L 18T 17

S5

Table S3. Catalytic activity of the mutant K524R and K524H toward the transformation of 1 to 3.

Enzymes Conv. (%)WT 14

K524R 7K524H 6

S6

Table S4. List of primers for saturation mutagenesis of single residue using NDT/VMA/ATG/TGG as degenerate codons.a

Primer mix Name Sequence (5’-3’)

F1

SrCAR-T265-265NDT-F AGTCTGCTGATCTATNDTAGTGGTAGTACCGGTSrCAR-T265-265VMA-F AGTCTGCTGATCTATVMAAGTGGTAGTACCGG

TSrCAR-T265-265ATG-F AGTCTGCTGATCTATATGAGTGGTAGTACCGGTSrCAR-T265-265TGG-F AGTCTGCTGATCTATTGGAGTGGTAGTACCGG

T

F2

SrCAR-S266-266NDT-F CTGCTGATCTATACCNDTGGTAGTACCGGTACA

SrCAR-S266-266VMA-F CTGCTGATCTATACCVMAGGTAGTACCGGTACA

SrCAR-S266-266ATG-F CTGCTGATCTATACCATGGGTAGTACCGGTACASrCAR-S266-266TGG-F CTGCTGATCTATACCTGGGGTAGTACCGGTAC

A

F3

SrCAR-G267-267NDT-F CTGATCTATACCAGTNDTAGTACCGGTACACCG

SrCAR-G267-267VMA-F

CTGATCTATACCAGTVMAAGTACCGGTACACCG

SrCAR-G267-267ATG-F CTGATCTATACCAGTATGAGTACCGGTACACCGSrCAR-G267-267TGG-F CTGATCTATACCAGTTGGAGTACCGGTACACC

G

F4

SrCAR-S408-408NDT-F ACCGCCGGCAGTGGTNDTGCCCCGATGAGCCCG

SrCAR-S408-408VMA-F ACCGCCGGCAGTGGTVMAGCCCCGATGAGCCCG

SrCAR-S408-408ATG-F ACCGCCGGCAGTGGTATGGCCCCGATGAGCCCG

SrCAR-S408-408TGG-F ACCGCCGGCAGTGGTTGGGCCCCGATGAGCCCG

F5

SrCAR-G430-430NDT-F GTGCATCTGGTGGATNDTTATGGTAGCACCGAA

SrCAR-G430-430VMA-F

GTGCATCTGGTGGATVMATATGGTAGCACCGAA

SrCAR-G430-430ATG-F GTGCATCTGGTGGATATGTATGGTAGCACCGAA

SrCAR-G430-430TGG-F GTGCATCTGGTGGATTGGTATGGTAGCACCGAA

F6

SrCAR-Y431-431NDT-F CATCTGGTGGATGGTNDTGGTAGCACCGAAGCC

SrCAR-Y431-431VMA-F CATCTGGTGGATGGTVMAGGTAGCACCGAAGCC

SrCAR-Y431-431ATG-F CATCTGGTGGATGGTATGGGTAGCACCGAAGCC

SrCAR-Y431-431TGG-F CATCTGGTGGATGGTTGGGGTAGCACCGAAGCC

F7

SrCAR-G432-432NDT-F CTGGTGGATGGTTATNDTAGCACCGAAGCCGGT

SrCAR-G432-432VMA-F

CTGGTGGATGGTTATVMAAGCACCGAAGCCGGT

SrCAR-G432-432ATG-F CTGGTGGATGGTTATATGAGCACCGAAGCCGGT

SrCAR-G432-432TGG-F CTGGTGGATGGTTATTGGAGCACCGAAGCCGGT

F8 SrCAR-T434-434NDT-F GATGGTTATGGTAGCNDTGAAGCCGGTCCGGTG

SrCAR-T434-434VMA-F GATGGTTATGGTAGCVMAGAAGCCGGTCCGG

S7

TGSrCAR-T434-434ATG-F GATGGTTATGGTAGCATGGAAGCCGGTCCGGT

GSrCAR-T434-434TGG-F GATGGTTATGGTAGCTGGGAAGCCGGTCCGG

TG

F9

SrCAR-T505-505NDT-F GATGGCTTTTATCTGNDTGGCGATGTTGTTGCC

SrCAR-T505-505VMA-F GATGGCTTTTATCTGVMAGGCGATGTTGTTGCC

SrCAR-T505-505ATG-F GATGGCTTTTATCTGATGGGCGATGTTGTTGCC

SrCAR-T505-505TGG-F GATGGCTTTTATCTGTGGGGCGATGTTGTTGCC

F10

SrCAR-D507-507NDT-F TTTTATCTGACCGGCNDTGTTGTTGCCGAAGTG

SrCAR-D507-507VMA-F TTTTATCTGACCGGCVMAGTTGTTGCCGAAGTG

SrCAR-D507-507ATG-F TTTTATCTGACCGGCATGGTTGTTGCCGAAGTG

SrCAR-D507-507TGG-F TTTTATCTGACCGGCTGGGTTGTTGCCGAAGTG

F11

SrCAR-Y519-519NDT-F CCGGAAGAATTTGTGNDTGTTGATCGTCGTAAA

SrCAR-Y519-519VMA-F CCGGAAGAATTTGTGVMAGTTGATCGTCGTAAA

SrCAR-Y519-519ATG-F CCGGAAGAATTTGTGATGGTTGATCGTCGTAAA

SrCAR-Y519-519TGG-F CCGGAAGAATTTGTGTGGGTTGATCGTCGTAAA

F12SrCAR-R522-522NDT-F TTTGTGTATGTTGATNDTCGTAAAAATGTTCTGSrCAR-R522-522VMA-F TTTGTGTATGTTGATVMACGTAAAAATGTTCTGSrCAR-R522-522ATG-F TTTGTGTATGTTGATATGCGTAAAAATGTTCTGSrCAR-R522-522TGG-F TTTGTGTATGTTGATTGGCGTAAAAATGTTCTG

F13

SrCAR-K524-524NDT-F TATGTTGATCGTCGTNDTAATGTTCTGAAACTGSrCAR-K524-524VMA-F TATGTTGATCGTCGTVMAAATGTTCTGAAACT

GSrCAR-K524-524ATG-F TATGTTGATCGTCGTATGAATGTTCTGAAACTGSrCAR-K524-524TGG-F TATGTTGATCGTCGTTGGAATGTTCTGAAACTG

F14

SrCAR-V936-936NDT-F ACCTATCTGAGTACCNDTGCCGTGGCAGTGGGC

SrCAR-V936-936VMA-F ACCTATCTGAGTACCVMAGCCGTGGCAGTGGGC

SrCAR-V936-936ATG-F ACCTATCTGAGTACCATGGCCGTGGCAGTGGGC

SrCAR-V936-936TGG-F ACCTATCTGAGTACCTGGGCCGTGGCAGTGGGC

F15

SrCAR-A937-937NDT-F TATCTGAGTACCGTGNDTGTGGCAGTGGGCGTG

SrCAR-A937-937VMA-F TATCTGAGTACCGTGVMAGTGGCAGTGGGCGTG

SrCAR-A937-937ATG-F TATCTGAGTACCGTGATGGTGGCAGTGGGCGTG

SrCAR-A937-937TGG-F TATCTGAGTACCGTGTGGGTGGCAGTGGGCGTG

F16

SrCAR-M999-999NDT-F GTGTTTCGTAGTGATNDTATTCTGGCACATCGCSrCAR-M999-999VMA-F

GTGTTTCGTAGTGATVMAATTCTGGCACATCGC

SrCAR-M999-999TGG-F

GTGTTTCGTAGTGATTGGATTCTGGCACATCGC

F17 SrCAR-Q1015- CTGAATGTGCCGGATNDTTTTACCCGCCTGAT

S8

1015NDT-F TSrCAR-Q1015-1015VMA-F

CTGAATGTGCCGGATVMATTTACCCGCCTGATT

SrCAR-Q1015-1015ATG-F

CTGAATGTGCCGGATATGTTTACCCGCCTGATT

SrCAR-Q1015-1015TGG-F

CTGAATGTGCCGGATTGGTTTACCCGCCTGATT

a NDT refers to Asparagine(N), Serine(S), Isoleucine(I), Histidine(H), Arginine(R), Leucine(L), Tyrosine(Y), Cysteine(C), Phenylalanine(F), Aspartic acid(D), Glycine(G), and Valine(V). VMA refers to Glutamic acid(E), Alanine(A), Glutamine(Q), Proline(P), Lysine(K), and Threonine(T). ATG refer to Methionine(M). TGG refer to Tryptophan(W).

S9

Supporting figures

Figure S1. The conversion of SrCAR mutants K524W and K524W/A937V in the reaction of benzoic acid in 0.5 h and 1 h reaction time. Error bars depict standard deviations over the 3 independent replicates. The reaction was analyzed by HPLC using an Agilent ZORBAX SB C18 column (4.6 mm × 250 mm × 5 μm) at a flow rate of 1 mL/min (the ratio of acetonitrile to 0.1% TFA was 3:7) and detection at 258 nm.

S10

Figure S2. Consensus analysis of SrCAR (PDB ID 5MST) with other crystallized ANL family members. The residues T265, G267, Y431, G432, T434 and D507 are indicated by red arrows. The figure was prepared by ESPript 3.0 (Robert and Gouet, 2014), using PDB database and E-value was set as 1e-6.

S11

Figure S3. GC profiles of WT and the two mutants. Compounds and corresponding peaks are indicated by black arrows.

S12

Figure S4. Michaelis-Menton curve fit of the wildtype of SrCAR (A) and the mutant K524Q (B), K524W (C), K524A (D), K524C (E), K524L (F), K524M (G) and K524V (H) in the reduction of benzoic acid.

S13

Figure S5. Comparison of biotransformation 1 with formation of 3 employing WT, K524Q and K524W with 20 mM substrate loadings.

S14

Figure S6. The position 524 (colored in magenta) is located at the surface of SrCAR, observed both in the adenylation (A) and thiolation (B) conformations.

S15

Figure S7. Traces of selected distances (d1 and d2) monitored in the MD runs of the systems regarding to WT (A), K524Q (B) and K524W (C). AMP is colored in blue, while the side chain of residue 524 located at the A domain is displayed in black.

S16

Figure S8. Determination of dissociation constant (Kd) of AMP (A) and PPi (B) for wild-type and variant K524W by ITC. The ITC experiments were performed using a MicroCal ITC titration calorimeter (ITC200, USA). Samples were prepared in titration buffer containing 20 mM PBS (pH 7.4) and 0.1 M NaCl. The reaction cell contained a degassed solution of 25 μM purified enzyme mixed with 13 x 2 μL aliquots of 750 μM AMP and PPi, respectively.

S17

Figure S9. Root mean square deviations (RMSD) measured during 300 ns MD simulations for models WT (A), K524Q (B) and K524W (C).

S18

References

Gulick, A.M., 2009. Conformational dynamics in the acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS. Chem. Biol. 4, 811-827. https://doi.org/10.1021/cb900156h.

Robert, X., Gouet, P., 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42 (W1), 320-324. https://doi.org/10.1093/nar/gku316.

Stolterfoht, H., Steinkellner, G., Schwendenwein, D., Pavkov-Keller, T., Gruber, K., Winkler, M., 2018. Identification of key residues for enzymatic carboxylate reduction. Front. Microbiol. 9, 250. https://doi.org/10.3389/fmicb.2018.00250.

Stolterfoht, H., Schwendenwein, D., Sensen, C.W., Rudroff, F., Winkler, M., 2017. Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes. J. Biotechnol. 257, 222-232. https://doi.org/10.1016/j.jbiotec.2017.02.014.

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