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Substrate specificity, regiospecificity and processivity in Glycoside Hydrolase family 74

Gregory Arnal1, Peter J. Stogios2, Jathavan Asohan1, Mohamed Attia1, Tatiana Skarina2, Alexander Holm

Viborg1, Bernard Henrissat3,4, Alexei Savchenko2,5,*, Harry Brumer,1,*.

List of the material included:

Table S1: Primers used for the PCR amplification of GH74 modules encoding sequence.

Table S2: Primers used for the site-directed mutagenesis of PoGH74cat and CjGH74.

Table S3: X-ray crystallographic statistics.

Figure S1: pH profiles.

Figure S2: temperature profiles

Figure S3: Michael Menten kinetics

Figure S4: Time course analysis of tamarind XyG hydrolysis in early stage of the reaction by

HPAEC-PAD

Figure S5: Protein sequence alignment of PoGH74, PgGH74 and SrGh74 from group 5,

SvGH74b from group 4, SatGH74 from Group3 and ClGH74a and NkGH74 from Group1.

Figure S6: Investigation of Cellvibrio japonicus GH74 mode of action.

Figure S7. Regiospecificity of PoGH74 wild type, of ΔAsn642-Asn651 variant and of different

point mutations of residue G442 located in the (-1) subsite of the active cleft of PoGH74.

Figure S8: Details of active sites of PgGH74 and PoGH74cat•(XXLG+XGXXLG) (PDB 6MGL)

crystal structures.

Supplementary file: GH74-bioinformatics analysis.mfa. This file contains the alignment used for

generating the phylogenetic tree in the Figure 1 in the main text. It contains all the non-redundant

sequences encoding for GH74 catalytic modules referenced in the CAZy database as well as the

sequence of the previously characterized GH74 modules from Xanthomonas citri pv.

mangiferaeindicae (Genbank accession number CCG35167) and Aspergillus fumigatus (Genbank

XP_747057), and the sequence of three uncharacterized GH74 modules from Paenibacillus

polymyxa Sb3-1, Paenibacillus jamilae and Streptomyces atroolivaceus (Genbank

WP_019687396, WP_063210590 and WP_033303664, respectively) that were recombinantly

produced in this study.

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Table S1: Primers used for the PCR amplification of GH74 modules encoding sequence. LIC-compatible extensions

TACTTCCAATCCAATGCCATG and TTATCCACTTCCAATGTTA were added at the 5’ extremity of each forward and reverse primers,

respectively.

Protein

(GenBank)

Bacterial strain

(DSM-No) Primer forward Primer reverse

Cloning vector-

N-terminal protein tag

ClGH74a

(AEM74844)

Caldicellulosiruptor lactoaceticus

(DSM 9545)

GCAGCGTCGGAACCATAC

ACATGG

AGAGGCTGTTGGTGTTGGC

AGTCC pMCSG53-His

ClGH74b

(AEM72711)

Caldicellulosiruptor lactoaceticus

(DSM 9545)

GCTGAAGTTCTTTCACAAA

AATATGTATGGAAGAATG

TGTTACTGTTCCACCACTTC

CACTATCAATTTC pMCSG69-MBP

CbGH74

(ACM60948)

Caldicellulosiruptor bescii

(DSM 6725)

AGTATAGTCACCACTCAAA

AATACATTTGGAAAAATG

TGATGTTGGAATTGTAGCA

CTATCAGCAATTTC pMCSG69-MBP

NkGH74

(AEV98114)

Niastella koreensis GR20-10

(DSM 17620)

ACAAAACTAATTGCACAA

ACATTTGGTAATGTAGCC

ATGGGTAAATTGAAGGGG

CAGCGC pMCSG53-His

PgGH74

(AIQ68396)

Paenibacillus graminis

(DSM 15220)

AGCGAAGCCTATAACTGG

AAAAGTGTCG

CACTGGATCGGCATAGAGC

GTGC pMCSG53-His

PbGH74

(AIQ58082)

Paenibacillus borealis

(DSM 13188)

AGTGAAGCCTACAACTGG

AACAGCG

TACCGGATCGGCGTATAAG

GTACC pMCSG53-His

PpGH74

(WP_019687396)

Paenibacillus polymyxa

(DSM 36)

GCTCCAAGTGACGATTATA

CTTGGAAAAG

TTACACACGATCTGCCACC

AGAATGCC pMCSG53-His

PjGH74

(WP_063210590)

Paenibacillus jamilae

(DSM13815)

GCTCCGAGTGACGATTATA

CTTGGAAAAG

TTATACACGATCTGCCACC

AGAATGCC pMCSG53-His

PmGH74

(AFC30493)

Paenibacillus mucilaginosus

(synthetic gene)

GTGAAAACACAAGCGTAC

GACTGGG

ATCCTTCATGTCCCCGCGA

ACG pMCSG53-His

RaGH74a

(ADU23213)

Ruminococcus albus 7

(DSM 20455)

GCTCAGGATATTTCATCGT

CTATGGAATGG

TGTTATCTTGGGAGCCTTG

ATATCTCCG pMCSG-GST

RaGH74b

(ADU20593)

Ruminococcus albus 7

(DSM 20455)

TCTGTTGATACGGGCTGGG

AATGG

TCACTTAACATCTCCACGG

ATTATACCGC pMCSG53-His

AFV00434 Simiduia agarivorans SA1

(DSM 21679)

ATGGCCGCTACCGTATGGC

AGC

CGCCGGATCCATATTCATG

CCGC pMCSG69-MBP

AFV00474 Simiduia agarivorans SA1

(DSM 21679)

ACACCCAATTGGGAATCAC

TCAATCCC

CGACAGACCATCAGGCGCC

G pMCSG69-MBP

SrGH74

(AGP53770)

Streptomyces rapamycinicus

(DSM 41530)

GCCGACGCCTACACCTGGA

AG

TCAGGAGATGTCGCCGCGC

AG pMCSG53-His

SvGH74a

(CCA60033)

Streptomyces venezuelae

(DSM 40230) GCGGCGACCGGCCCGTAC

TCAGGACGGGTCGCCGTAC

TG pMCSG53-His

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SvGH74b

(CCA55868)

Streptomyces venezuelae

(DSM 40230)

CCCTCCTACACCTGGAAGA

ACGTC CTGCTCGGCCGTGTCGCC pMCSG53-His

SatGH74

(WP_033303664)

Streptomyces atroolivaceus

(DSM 40137)

GCGAAGACCCAGCCGCAC

CACTGG

CTACTGGGGGTCTGCGTAC

TGG pMCSG53-His

Table S2: Primers used for the site-directed mutagenesis of PoGH74cat and CjGH74. Bold characters indicate point mutations.

Protein Matrix DNA Primer forward Primer reverse Reference

PoGH74cat-G476A pMCSG53::PoGH74cat CACTGCACTTGCCGACGT

TTCCGGTTTC

GAAACCGGAAACGTCGG

CAAGTGCAGTG

Arnal et al.

PoGH74cat-G476Q pMCSG53::PoGH74cat CACTGCACTTCAAGACGT

TTCCGGTTTC

GAAACCGGAAACGTCTT

GAAGTGCAGTG

Arnal et al.

PoGH74cat-G476W pMCSG53::PoGH74cat CACTGCACTTTGGGACGT

TTCCGGTTTC

GAAACCGGAAACGTCCC

TAAGTGCAGTG

Arnal et al.

CjGH74-W353A pET28a::CjGH74 GCGTCGCAAATTGCCGCG

TGGCCCGATGCGGTT

AACCGCATCGGGCCACG

CGGCAATTTGCGACGC

Attia et al.

CjGH74-W354A pET28a::CjGH74 TCGCAAATTGCCTGGGCG

CCCGATGCGGTTTTC

GAAAACCGCATCGGGCG

CCCAGGCAATTTGCGA

Attia et al.

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Table S3: X-ray crystallographic statistics

Structure AFV00434 NkGH74•(XXLG/XXXG) ClGH74a•(LLG) PgGH74 SrGH74•(XLLG/XXXG)

PDB code 6P2K 6P2L 6P2M 6P2N 6P2O

Data collection

Space group P213 P22121 P212121 P212121 P212121

Unit cell a, b, c (Å) α, β, γ, (°)

173.4, 173.4, 173.4 90, 90, 90

42.9, 119.1, 127.0 90, 90, 90

65.0, 72.8, 150.7 90, 90, 90

57.5, 86.2, 130.2 90, 90, 90

93.0, 112.8, 222.8 90, 90, 90

Resolution, Å 40.0 – 2.15 30.0 – 1.08 25.00 – 1.98 35.00 – 1.35 25.00 – 1.88 Rmerge

a

Rpimb

0.072 (0.849)c 0.032 (0.441)

0.073 (0.825) 0.037 (0.447)

0.165 (0.873) 0.063 (0.438)

0.049 (0.838) 0.021 (0.449)

0.082 (0.954) 0.043 (0.565)

CC1/2c 0.676 0.605 0.623 0.567 0.541

I / (I) 22.2 (1.8) 25.9 (1.7) 18.2 (2.4) 31.0 (1.7) 12.2 (1.1)

Completeness, % 99.8 (97.5) 98.3 (96.0) 100 (99.9) 96.1 (96.8) 89.4 (90.1) Redundancy 5.7 (4.6) 4.5 (4.1) 7.8 (6.3) 6.1 (4.2) 3.7 (3.6) Refinement Resolution, Å 38.78 – 2.15 29.3 – 1.08 24.6 – 1.98 30.5 – 1.35 25.0 – 1.88 No. unique reflections: working, test

93430, 4654 262889, 1916 47656, 1875 132023, 3636

156964, 3304

R-factor/free R-factord 15.4/20.4 (23.8/27.4)

11.9/14.0 (23.9/24.5) 15.6/20.4 (23.8/25.6)

14.9/18.0 (22.9/20.8)

14.8/18.0 (28.9/30.4)

No. refined atoms Protein Carbohydrate Solvent Water

12080 N/A 150 1794

10165 155 27 1562

5294 74 45 1229

5724 N/A 21 1339

10995 381 40 2662

B-factors Protein Carbohydrate Solvent Water

33.8 N/A 68.8 44.3

11.4 22.7 17.6 31.8

23.3 47.5 52.1 39.8

17.3 N/A 34.2 33.4

23.3 35.6 66.8 41.6

r.m.s.d. Bond lengths, Å

Bond angles,

0.004 0.663

0.008 1.112

0.003 0.572

0.013 1.289

0.013 1.133

Ramachandran favoured 1501/1556 (96%) 690/711 (97%) 682/700 (97%) 731/761 (96%) 1402/1460 (96%) allowed 53/1556 (3%) 21/711 (3%) 18/700 (3%) 30/761 (4%) 58/1460 (4%) outliers 2/1556 (0%) 0 0 0 0

aRmerge = hklj|Ihkl.j - Ihkl|/hkljIhk,j, where Ihkl,j and Ihkl are the jth and mean measurement of the

intensity of reflection j. bRpim = hkl√(n/n-1) n

j=1|Ihkl.j - Ihkl|/hkljIhk,j call values in brackets and CC1/2 refer to the highest resolution shell. dR = |Fp

obs – Fpcalc|/Fp

obs, where Fpobs and Fp

calc are the observed and calculated structure factor

amplitudes, respectively.

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Figure S1: pH profiles for A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a (Group 1), D-

SatGH74 (Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5), H-RaGH74b

(Group 5), I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74 (Group 5), M-

PpGH74 (Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5). Blue circles for citrate buffer; Green

crosses for phosphate buffer. Error bars represent the standard deviation for three replicates.

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Figure S2: temperature profiles for A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a (Group

1), D-SatGH74 (Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5), H-

RaGH74b (Group 5), I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74

(Group 5), M-PpGH74 (Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5). Error bars represent

the standard deviation for three replicates.

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Figure S3: Michael Menten kinetics for A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a

(Group 1), D-SatGH74 (Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5),

H-RaGH74b (Group 5), I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74

(Group 5), M-PpGH74 (Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5). Error bars represent

the standard deviation for three replicates.

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Figure S4: Time course analysis of tamarind XyG hydrolysis in early stage of the reaction by

HPAEC-PAD. A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a (Group 1), D-SatGH74

(Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5), H-RaGH74b (Group 5),

I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74 (Group 5), M-PpGH74

(Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5)

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Figure S5: Protein sequence alignment of PoGH74, PgGH74 and SrGh74 from group 5, SvGH74b

from group 4, SatGH74 from Group3 and ClGH74a and NkGH74 from Group1. Stars indicate active cleft

hydrophobic residues and their subsite position. The color code of the stars corresponds to their

conservation across the phylogenetic groups: blue stars when the residue is only conserved in Group 5,

purple stars when the residue is conserved in Groups 4 and 5, green stars when the residue is conserved in

Groups 3, 4 and 5, red stars when the residue is conserved across all Groups. Red triangles indicate the

catalytic residues. Grey diamond refers to the residue in the -1 subsite dictating regiospecificity. Green

bars indicate specific loop extensions. Residue numbering is based on PoGH74 full-length protein.

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Figure S6: Investigation of Cellvibrio japonicus GH74 mode of action. HPAEC-PAD analysis of time-

course tamarind XyG hydrolysis by A. CjGH74 wild type, B. CjGH74-W353A variant (+3 subsite) and

C. CjGH74-W354A variant (+5 subsite). The same stock solution of tamarind XyG was used for each

variant, hence all “no enzyme” traces are identical. The same reference chromatogram of xyloglucan

oligosaccharide standards is presented for comparison with the products of each variant.

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Figure S7. Regiospecificity of PoGH74 wild type, of ΔAsn642-Asn651 variant and of different point

mutations of residue G442 located in the (-1) subsite of the active cleft of PoGH74. Limit digest of

(A) XXXGXXXG and (B) XXXG analyzed by HPAEC-PAD.

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Figure S8: Details of active sites of PgGH74 and PoGH74cat•(XXLG+XGXXLG) (PDB 6MGL) crystal

structures. Comparison of PgGH74 and PoGH74cat•(XXLG+XGXXLG) (PDB 6MGL), showing the

presence of 12 aromatic residues in the active site cleft of PgGH74 and conformational differences in the -

4, -3 and -2 subsites (i.e. different positions of W126, Y214 and Y122 of PgGH74 relative to their

equivalents in PoGH74 – W91, Y87, Y219).