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Glycoside Hydrolase Family 10
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|Glycoside Hydrolase Family GH10|
|Active site residues||known|
|CAZy DB link|
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  and follow a classical Koshland double-displacement mechanism. Enzymes that have been well-studied kinetically include the Cellulomonas fimi endo-glycanase (Cex)*, for which a detailed kinetic study involving both steady state and pre-steady state kinetic analyses was performed . Recent studies of the roles of each substrate hydroxyl in catalysis have also been described . Detailed analyses of substrate and subsite specificities of the Pseudomonas cellulosa xylanase have also been described .
* 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 . 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].
The catalytic nucleophile was first identified in the Cellulomonas fimi endo-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 . 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 . 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 are available for a large number of Family GH10 enzymes, the first solved being those of the Streptomyces lividans xylanase A , the C. fimi endo-glycanase Cex , and the Cellvibrio japonicus Xyn10A (previously Pseudomonas fluorescens subsp. xylanase A) . 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) .
- First sterochemistry determination
- Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by NMR 
- First catalytic nucleophile identification
- Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by 2-fluoroglucose labeling 
- First general acid/base residue identification
- Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by rescue kinetics with mutants 
- First 3-dimensional structure
- Cellulomonas fimi endo-xylanase Cex (CfXyn10A) , Streptomyces lividans xylanase (SlXyn10A) , and Cellvibrio japonicus Xyn10A (previously Pseudomonas fluorescens subsp. xylanase A) .
- Withers SG, Dombroski D, Berven LA, Kilburn DG, Miller RC Jr, Warren RA, and Gilkes NR. Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex. Biochem Biophys Res Commun. 1986 Sep 14;139(2):487-94.
- Tull D and Withers SG. Mechanisms of cellulases and xylanases: a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi. Biochemistry. 1994 May 24;33(20):6363-70.
- Wicki J, Schloegl J, Tarling CA, and Withers SG. Recruitment of both uniform and differential binding energy in enzymatic catalysis: xylanases from families 10 and 11. Biochemistry. 2007 Jun 12;46(23):6996-7005. DOI:10.1021/bi700359e |
- Andrews SR, Charnock SJ, Lakey JH, Davies GJ, Claeyssens M, Nerinckx W, Underwood M, Sinnott ML, Warren RA, and Gilbert HJ. Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A. J Biol Chem. 2000 Jul 28;275(30):23027-33. DOI:10.1074/jbc.M000128200 |
- Henrissat B, Teeri TT, and Warren RA. A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. FEBS Lett. 1998 Mar 27;425(2):352-4.
- Henrissat B, Claeyssens M, Tomme P, Lemesle L, and Mornon JP. Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989 Sep 1;81(1):83-95.
- Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, and Warren RA. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303-15.
- Tull D, Withers SG, Gilkes NR, Kilburn DG, Warren RA, and Aebersold R. Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi. J Biol Chem. 1991 Aug 25;266(24):15621-5.
- MacLeod AM, Lindhorst T, Withers SG, and Warren RA. The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants. Biochemistry. 1994 May 24;33(20):6371-6.
- Derewenda U, Swenson L, Green R, Wei Y, Morosoli R, Shareck F, Kluepfel D, and Derewenda ZS. Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1,4-D-glycanases. J Biol Chem. 1994 Aug 19;269(33):20811-4.
- White A, Withers SG, Gilkes NR, and Rose DR. Crystal structure of the catalytic domain of the beta-1,4-glycanase cex from Cellulomonas fimi. Biochemistry. 1994 Oct 25;33(42):12546-52.
- Harris GW, Jenkins JA, Connerton I, Cummings N, Lo Leggio L, Scott M, Hazlewood GP, Laurie JI, Gilbert HJ, and Pickersgill RW. Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites. Structure. 1994 Nov 15;2(11):1107-16.
- Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7090-4.