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Although a few glycoside hydrolases of this family show endo-beta-1,3-xylanase activity, the majority of the enzymes are endo-beta-1,4-xylanases. Some of the latter display limited activity on aryl cellobiosides, but not on cellulose.
Kinetics and Mechanism
Family GH10 xylanases are retaining enzymes, as first shown by NMR [1] and follow a classical Koshland double-displacement mechanism. Enzymes that have been well-studied kinetically include the Cellulomonas fimiendo-glycanase (Cex)*, for which a detailed kinetic study involving both steady state and pre-steady state kinetic analyses was performed [2]. Recent studies of the roles of each substrate hydroxyl in catalysis have also been described [3]. Detailed analyses of substrate and subsite specificities of the Pseudomonas cellulosa xylanase have also been described [4].
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* This enzyme has frequently (and erroneously) been called exo-cellulase in the literature (hence the name Cex). The error came from the low, but significant activity of the enzyme on aryl-beta-cellobioside, a substrate once thought to be specific of exo-cellulases. Although everyone agrees now that this is an endo-1,4-xylanase, the Vancouver group still calls it Cex "endo-glycanase" instead of calling it xylanase CfXyn10A [5]. Indeed, prior to extensive biochemical analysis, GH10 was one of the first glycoside hydrolase families classified by hydrophobic cluster analysis, and was previously known as "Cellulase Family F" [6, 7].
Catalytic Residues
The catalytic nucleophile was first identified in the Cellulomonas fimiendo-xylanase (CfXyn10A) as Glu233 (earlier numbered as 274) in the sequence ITELD through trapping of the 2-deoxy-2-fluoroglucosyl-enzyme intermediate and subsequent peptide mapping [8]. The general acid/base residue was first identified as Glu127 in this same enzyme through detailed mechanistic analysis of mutants at that position, which included azide rescue experiments [9]. Family GH10 enzymes, as is typical of Clan GHA, have an asparagine residue preceding the general acid/base residue in a typical NEP sequence. The asparagine engages in important hydrogen-bonding interactions with the substrate 2-hydroxyl.
Three-dimensional structures
Three-dimensional structures are available for a large number of Family GH10 enzymes, the first solved being those of the Streptomyces lividans xylanase A [10], the C. fimi endo-glycanase Cex [11], and the Cellvibrio japonicus Xyn10A (previously Pseudomonas fluorescens subsp. xylanase A) [12]. As members of Clan GHA they have a classical (α/β)8 TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of beta-strands 4 (acid/base) and 7 (nucleophile) [13].
Family Firsts
First sterochemistry determination
Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by NMR [1]
