Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 <i>endo</i>-xyloglucanase

Arnal, Grégory; Stogios, P.J.; Asohan, Jathavan; Skarina, T.; Savchenko, Alexei; Brumer, Harry

doi:10.1042/bcj20180763

Cited by 15 publications

(22 citation statements)

References 64 publications

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“…S3). These values are in the same range as previously characterized GH74 enzymes (16,22,29,35). Exceptionally, recombinant Streptomyces venezuelae GH74b was very unstable and precipitated rapidly in solution, which did not allow accurate kinetic characterization.…”

Section: Production and Biochemical Characterizationsupporting

confidence: 73%

“…The mode of action of GH74 xyloglucanases has been described for a limited number of enzymes. Oligoxyloglucan reducing end-specific cellobiohydrolases (OXG-RCBHs) ( EDITORS' PICK: GH74 structure-function analysis wide distribution of product chain lengths (23,26,33), or they can act in a processive fashion to rapidly release small xyloglucan oligosaccharides (XyGOs) at early stages of XyG hydrolysis (20,26,28,29,35). To investigate the mode of action of our recombinant GH74 enzymes, we analyzed the time-course hydrolysis of tamarind XyG at early stages of the reaction by HPAEC-PAD ( Fig.…”

Section: Regiospecificity and Processivity Of Gh74 Membersmentioning

confidence: 99%

“…S4). Also, most previously characterized GH74 endo-xyloglucanases release XXXG-type XyGOs via the exclusive hydrolysis of XyG at unbranched glucosyl residues (16,22,28,33,50); however, some cleave exclusively at xylosylated glucosyl units (21,23), and others can cleave at both G and X motifs (20,26,32,35). To investigate the backbone regiospecificity of GH74 enzymes, the limit digests of tamarind XyG, of XXXGXXXG, and of XXXG were analyzed by HPAEC-PAD (Fig.…”

Section: Regiospecificity and Processivity Of Gh74 Membersmentioning

confidence: 99%

“…This structure validates the HCA prediction of a distant relationship to GH74 and suggests a superfamily or "clan" (53). Two aspartic acid residues (Asp 35 and Asp 419 ), located 8.3 Å apart in the putative catalytic site, have an adequate spatial position to catalyze the hydrolysis of the glycosidic bond via an inverting mechanism. However, comparison of the protein backbone in the crystal structure of AFV00434 and Group 5 Paenibacillus odorifer GH74 (PDB entry 6MGL) (35) revealed that AFV00434 loops Trp 121 -Ala 136 , Ser 468 -Asn 476 , and Ser 706 -Tyr 716 obstruct the active cleft at subsites Ϫ3/Ϫ2, ϩ1/ϩ2, and Ϫ4/Ϫ3, respectively, providing a possible explanation for the lack of polysaccharide hydrolysis (Fig.…”

Section: Regiospecificity and Processivity Of Gh74 Membersmentioning

confidence: 99%

“…Endoxyloglucanases can be further delineated into endo-dissociative enzymes, which hydrolyze the backbone and immediately release both new chain ends, and endo-processive enzymes, which perform multiple hydrolytic events, releasing short oligosaccharides before disengaging. The ability of some GH74 enzymes (26,28,29,35) to act processively on soluble XyG is notable, considering that this mode of action is more commonly associated with GHs acting on crystalline cellulose or chitin (42)(43)(44)(45)(46)(47).…”

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Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Arnal

Stogios

Asohan

et al. 2019

Journal of Biological Chemistry

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Glycoside hydrolase family 74 (GH74) is a historically important family of endo-␤-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: exo, endo-dissociative, and endo-processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures, as well as one distantly related dual seven-bladed ␤propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecificity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies. Terrestrial plants harbor ϳ80% of the biomass on Earth, some 450 gigatons of carbon, in the form of lignocellulose (cell walls comprised of cellulose, matrix glycans, lignin, and other polymers) (1). Although terrestrial biomass represents an attractive renewable resource for the production of fuels, chemicals, and materials for human consumption, the controlled degradation of lignocellulose, whether (thermo)chemical or enzymatic, is hindered by its heterogeneous composition and complex organization (2). Hence, significant efforts have been made to identify enzymes able to efficiently modify and deconstruct this complex material. Xyloglucans (XyGs) 3 comprise a prominent family of cell wall matrix glycans (hemicelluloses). XyGs are ubiquitous in land plants, in which they constitute up to 20% of the dry weight of cell walls (3, 4). Notably, XyGs are secreted by roots of diverse plant species and are therefore likely to actively influence rhizobiota (5). XyGs are also found as storage polysaccharides comprising ϳ50% of the mass of some seeds (e.g. tamarind and nasturtium) and therefore represent important agricultural byproducts with applications in the food, biomaterial, and medical sectors (6, 7). XyGs have a ␤-1,4-linked glucosyl backbone ("G" unit), some of which a...

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Section: Production and Biochemical Characterizationsupporting

confidence: 73%