Literature summary for 3.2.1.151 extracted from

Arnal, G.; Stogios, P.J.; Asohan, J.; Skarina, T.; Savchenko, A.; Brumer, H.
Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase (2018), Biochem. J., 475, 3963-3978 .

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Cloned(Commentary)

Cloned (Comment)	Organism
DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant catalytic domains in Escherichia coli strain BL21(DE3)	Paenibacillus odorifer
recombinant expression of His-tagged wild-type and mutant catalytic PoGH74cat enzyme modules in Escherichia coli strain BL21(DE3)	Paenibacillus odorifer

Crystallization (Commentary)

Crystallization (Comment)	Organism
PoGH74cat as apoenzyme and as XLX complex, sitting drop vapour diffusion method, 0.0015 ml of 20 mg/ml protein with 10 mM xylose and 0.1 M glucose are mixed with 0.0015 ml of the reservoir solution 25% w/v PEG 8000, 100 mM Bis-Tris buffer, pH 6.9, 1 mM tris(2-carboxyethyl)phosphine, and 0.2 M sodium acetate, crystals of PoGH74cat in complex with XLX are grown by cocrystallization using 500 nl of 10 mg/ml protein plus 5 mM of a xyloglucan oligosaccharide mixture (XXXG, XLXG, XXLG, and XLLG) mixed with 500 nl of the reservoir solution 20% w/v PEG3350 and 0.2 M sodium potassium tartrate. Crystals of the PoGH74catD70A mutant in complex with XXLG and XGXXLG are obtained by cocrystallization of 500 nl of 10 mg/ml protein with a complex XyGO mixture mixed with 500 nl reservoir solution 25% w/v PEG3350, 0.2 M magnesium chloride, and 100 mM Tris buffer, pH 8.5, 22°C, X-ray diffraction structure determination and analysis at 1.50-2.10 A resolution, molecular replacement and modeling	Paenibacillus odorifer

Crystallization (Comment)

Organism

PoGH74cat as apoenzyme and as XLX complex, sitting drop vapour diffusion method, 0.0015 ml of 20 mg/ml protein with 10 mM xylose and 0.1 M glucose are mixed with 0.0015 ml of the reservoir solution 25% w/v PEG 8000, 100 mM Bis-Tris buffer, pH 6.9, 1 mM tris(2-carboxyethyl)phosphine, and 0.2 M sodium acetate, crystals of PoGH74cat in complex with XLX are grown by cocrystallization using 500 nl of 10 mg/ml protein plus 5 mM of a xyloglucan oligosaccharide mixture (XXXG, XLXG, XXLG, and XLLG) mixed with 500 nl of the reservoir solution 20% w/v PEG3350 and 0.2 M sodium potassium tartrate. Crystals of the PoGH74catD70A mutant in complex with XXLG and XGXXLG are obtained by cocrystallization of 500 nl of 10 mg/ml protein with a complex XyGO mixture mixed with 500 nl reservoir solution 25% w/v PEG3350, 0.2 M magnesium chloride, and 100 mM Tris buffer, pH 8.5, 22°C, X-ray diffraction structure determination and analysis at 1.50-2.10 A resolution, molecular replacement and modeling

Paenibacillus odorifer

Protein Variants

Protein Variants	Comment	Organism
D70A	site-directed mutagenesis in the PoGH74cat module at the catalytic base	Paenibacillus odorifer
D70A	site-directed mutagenesis, mutation of the catalytic base, the mutant turnover rate is similar to wild-type	Paenibacillus odorifer
G476Y	site-directed mutagenesis in the PoGH74cat module at the -1 subsite	Paenibacillus odorifer
G476Y	site-directed mutagenesis, mutation of the -1 subsite, the mutant turnover rate is similar to wild-type	Paenibacillus odorifer
additional information	generation of PoGH74cat(DELTA642-651) and PoGH74cat(DELTA671-675) truncated mutants	Paenibacillus odorifer
additional information	construction of truncated mutants, i.e. PoGH74cat(DELTA642-651) and PoGH74cat(DELTA671-675), the mutant turnover rate is similar to wild-type	Paenibacillus odorifer
W347A	site-directed mutagenesis in the PoGH74cat module at the +3 subsite	Paenibacillus odorifer
W347A	site-directed mutagenesis, mutation of the +3 subsite, kcat value is increased by 5fold compared to wild-type	Paenibacillus odorifer
W348A	site-directed mutagenesis in the PoGH74cat module at the +5 subsite	Paenibacillus odorifer
W348A	site-directed mutagenesis, mutation of the +5 subsite, the mutant turnover rate is similar to wild-type	Paenibacillus odorifer
W406A	site-directed mutagenesis in the PoGH74cat module at the +2 subsite	Paenibacillus odorifer
W406A	site-directed mutagenesis, mutation of the +2 subsite, the mutant turnover rate is similar to wild-type	Paenibacillus odorifer
Y372A	site-directed mutagenesis in the PoGH74cat module at the +6 subsite	Paenibacillus odorifer
Y372A	site-directed mutagenesis, mutation of the +6 subsite, the mutant turnover rate is similar to wild-type	Paenibacillus odorifer

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	Michaelis-Menten kinetics	Paenibacillus odorifer
additional information	-	additional information	Michaelis-Menten kinetics, Km values for wild-type and mutant enzymes are 0.04-0.23 mg/ml	Paenibacillus odorifer

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
additional information	Paenibacillus odorifer	endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units	?	-	?
additional information	Paenibacillus odorifer DSM 15391	endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units	?	-	?
xyloglucan + H2O	Paenibacillus odorifer	-	xyloglucan oligosaccharides	-	?
xyloglucan + H2O	Paenibacillus odorifer DSM 15391	-	xyloglucan oligosaccharides	-	?

Organism

Organism	UniProt	Comment	Textmining
Paenibacillus odorifer	A0A1R0YRH0	-	-
Paenibacillus odorifer	A0A1R0ZNW0	-	-
Paenibacillus odorifer DSM 15391	A0A1R0YRH0	-	-
Paenibacillus odorifer DSM 15391	A0A1R0ZNW0	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant catalytic PoGH74cat enzyme modules from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, dialysis, and gel filtration	Paenibacillus odorifer
recombinant N-terminally His6-tagged wild-type and mutant catalytic domains from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, ultrafiltration, and gel filtration	Paenibacillus odorifer

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units	Paenibacillus odorifer	?	-	?
additional information	residue Gly476 is uniquely responsible for the promiscuous xyloglucan backbone-cleaving activity of the GH74 module PoGH74cat	Paenibacillus odorifer	?	-	?
additional information	enzymatic assays are performed on AZCL-xyloglucan, release of soluble dye fragments from cross-linked AZCL-XyG is measured. Enzyme PoGH74 shows essentially exclusive specificity for xyloglucan (XyG), which is typical for all GH74 members	Paenibacillus odorifer	?	-	-
additional information	enzymatic assays are performed on AZCL-xyloglucan, release of soluble dye fragments from cross-linked AZCL-XyG is measured. Enzyme PoGH74 shows essentially exclusive specificity for xyloglucan (XyG), which is typical for all GH74 members	Paenibacillus odorifer DSM 15391	?	-	-
additional information	endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units	Paenibacillus odorifer DSM 15391	?	-	?
additional information	residue Gly476 is uniquely responsible for the promiscuous xyloglucan backbone-cleaving activity of the GH74 module PoGH74cat	Paenibacillus odorifer DSM 15391	?	-	?
xyloglucan + H2O	the GH74 module (PoGH74cat) reveals a highly specific, processive endo-xyloglucanase activity that can hydrolyze the polysaccharide backbone at both branched and unbranched positions	Paenibacillus odorifer	?	-	?
xyloglucan + H2O	the GH74 module (PoGH74cat) reveals a highly specific, processive endo-xyloglucanase activity that can hydrolyze the polysaccharide backbone at both branched and unbranched positions	Paenibacillus odorifer DSM 15391	?	-	?
xyloglucan + H2O	-	Paenibacillus odorifer	xyloglucan oligosaccharides	-	?
xyloglucan + H2O	xyloglucan from tamarind seeds, structural analysis of xyloglucan binding structure with PoGH74cat, and structural basis of the bond cleavage pattern of PoGH74cat, overview	Paenibacillus odorifer	xyloglucan oligosaccharides	-	?
xyloglucan + H2O	-	Paenibacillus odorifer DSM 15391	xyloglucan oligosaccharides	-	?
xyloglucan + H2O	xyloglucan from tamarind seeds, structural analysis of xyloglucan binding structure with PoGH74cat, and structural basis of the bond cleavage pattern of PoGH74cat, overview	Paenibacillus odorifer DSM 15391	xyloglucan oligosaccharides	-	?

Subunits

Subunits	Comment	Organism
More	mature PoGH74 is composed of an N-terminal GH74 catalytic module in-train with three modules of unknown function (X2 domain, PFAM03442, and a carbohydrate-binding module (CBM) family 3), modular architecture of the native Paenibacillus odorifer AIQ73809 gene product, and GH74 enzymes structure comparisons, overview	Paenibacillus odorifer

Synonyms

Synonyms	Comment	Organism
bacterial glycoside hydrolase family 74 endo-xyloglucanase	-	Paenibacillus odorifer
endo-xyloglucanase	-	Paenibacillus odorifer
PODO_RS11130	locus name	Paenibacillus odorifer
PoGH74	-	Paenibacillus odorifer

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Paenibacillus odorifer

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
25.3	-	xyloglucan	xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catG476Y	Paenibacillus odorifer
39.8	-	xyloglucan	xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant wild-type enzyme PoGH74	Paenibacillus odorifer
43.8	-	xyloglucan	xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catW406A	Paenibacillus odorifer
47.5	-	xyloglucan	xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catY372A	Paenibacillus odorifer
58.3	-	xyloglucan	xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catW348A	Paenibacillus odorifer
214.7	-	xyloglucan	xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catW347A	Paenibacillus odorifer

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Paenibacillus odorifer

General Information

General Information	Comment	Organism
evolution	Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Endo-xyloglucanases, which catalyze the cleavage of the xyloglucans (XyGs) backbone (EC 3.2.1.151), are currently found in glycoside hydrolase (GH) families GH5, GH9, GH12, GH16, GH44, and GH74 (in CAZy classification). Of these, family GH74 is distinguished by fewer sequence members, an essentially singular specificity for xyloglucans, and a characteristic tertiary structure comprised of two 7-bladed beta-propeller domains that form a large interfacial cleft to accommodate the bulky polysaccharide	Paenibacillus odorifer
evolution	the endo-xyloglucanase belongs to the bacterial glycoside hydrolase family 74, GH74. Endo-xyloglucanases, which catalyze the cleavage of the XyGs backbone (EC 3.2.1.151), are currently found in glycoside hydrolase (GH) families GH5, GH9, GH12, GH16, GH44, and GH74 in the carbohydrate-active enzymes (CAZy) classification. Of these, family GH74 is distinguished by fewer sequence members, an essentially singular specificity for XyGs, and a characteristic tertiary structure comprised of two 7-bladed beta-propeller domains that form a large interfacial cleft to accommodate the bulky polysaccharide. Structure-activity relationships among characterized GH74 members, including determinants of endo versus exo (EC 3.2.1.150) activity, have been reviewed	Paenibacillus odorifer
malfunction	replacement of catalytic Gly476 with Tyr, which is conserved in many GH74 members, results in exclusive hydrolysis of xyloglucan at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity	Paenibacillus odorifer
physiological function	endo-xyloglucanases from the GH74 family hydrolyze xyloglucans (XyGs). Enzymatic XyG hydrolysis increases cellulose digestibility, which underscores their structural importance in the plant cell wall. XyGs are also found as storage polysaccharides in some seeds, e.g. tamarind, and therefore represent important agricultural byproducts that have applications in the food, biomaterial and medical sectors. The endo-xyloglucanase can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units	Paenibacillus odorifer