Abstract
Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs-mrs.fr/CAZY/), their biochemical significance is unclear. The Cellvibrio japonicus GH10 xylanase CjXyn10C is a bi-modular enzyme comprising a GH10 catalytic module and a family 15 carbohydrate-binding module. The three-dimensional structure at 1.85 A, presented here, shows that the sequence joining the two modules is disordered, confirming that linker sequences in modular glycoside hydrolases are highly flexible. CjXyn10C hydrolyzes xylan at a rate similar to other previously described GH10 enzymes but displays very low activity against xylooligosaccharides. The poor activity on short substrates reflects weak binding at the -2 subsite of the enzyme. Comparison of CjXyn10C with other family GH10 enzymes reveals "polymorphisms" in the substrate binding cleft including a glutamate/glycine substitution at the -2 subsite and a tyrosine insertion in the -2/-3 glycone region of the substrate binding cleft, both of which contribute to the unusual properties of the enzyme. The CjXyn10C-substrate complex shows that Tyr-340 stacks against the xylose residue located at the -3 subsite, and the properties of Y340A support the view that this tyrosine plays a pivotal role in substrate binding at this location. The generic importance of using CjXyn10C as a template in predicting the biochemical properties of GH10 xylanases is discussed.
Highlights
Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere
X-ray crystallography has provided detailed insights into the structural basis for the catalytic activity displayed by glycoside hydrolases, there is a lack of information on the conformation
This report reveals that CjXyn10C, while exhibiting a similar capacity to hydrolyze xylan to other GH10 enzymes, displays unusually poor activity against xylooligosaccharides
Summary
4-O-MeGlcA, 4-O-methyl-D-glucuronic acid; CjXyn10A, C. japonicus xylanase 10A; CjXyn10C, C. japonicus xylanase 10C; CjXyn10C-m, truncated CjXyn10C consisting of the CBM15 linked to the catalytic module; CjXyn10C-GH10, catalytic module of CjXyn10C; CfXyn10A, C. fimi xylanase 10A; GH, glycoside hydrolase family; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose; PNPX2, 4-nitrophenyl--xylobioside; PNPX, 4-nitrophenyl--xyloside; PNPG2, 4-nitrophenyl--cellobioside; GX, glucuronoxylan; d.p., degree of polymerization; CBM, carbohydrate binding module; r.m.s., root mean square. GH10 contains a large number of proteins, and the threedimensional structural data information available for several members of this family, coupled with sequence comparisons, can be used to identify amino acid “polymorphisms” within the substrate binding cleft of this family of enzymes. Examples of these variations include the substitution of a glutamate in the Ϫ2 subsite (which makes an important hydrogen bond with the O2 of xylose) with glycine in ϳ16% of GH10 enzymes. Dissecting the mechanistic implications of binding cleft variations will facilitate the use of a bioinformatics approach for predicting the biochemical properties of the extensive repertoire of glycoside hydrolases identified through genome sequencing programs
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