Glycoside Hydrolase Family 10

Glycoside Hydrolase Family GH10
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH10.html

Substrate specificities

Although a few members 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 fimi endo-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 exocellulases. 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.

Catalytic Residues

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 [5]. The acid/base catalyst was first identified as Glu127 in this same enzyme through detailed mechanistic analysis of mutants at that position, which included azide rescue experiments [6]. Family GH10 enzymes, as is typical of Clan GHA, have an asparagine residue preceding the acid/base catalyst 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 [7] and the C. fimi endo-glycanase Cex [8]. 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) [9].

PICTURES? OVERALL STRUCTURE; ACTIVE SITE; CARTOON OF ACTIVE SITE SHOWING INTERACTIONS?

For example:

• 3-D Structure of Bruce Stone:

• Link to a XET structure picture:
  • http://www.rcsb.org/pdb/explore/explore.do?structureId=1UMZ

Family Firsts

First sterochemistry determination

Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by NMR [1]

First nuc. ID

Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by 2-fluoroglucose labeling [5]

First A/B ID

Cellulomonas fimi endo-xylanase Cex (CfXyn10A) by rescue kinetics with mutants [6]

First 3-D structures of a (major subfamily)

Cellulomonas fimi endo-xylanase Cex (CfXyn10A) [8] and Streptomyces lividans xylanase (SlXyn10A) [7]

References

1. Withers SG, Dombroski D, Berven LA, Kilburn DG, Miller RC Jr, Warren RA, and Gilkes NR. (1986). 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;139(2):487-94. DOI:10.1016/s0006-291x(86)80017-1 |
2. Tull D and Withers SG. (1994). Mechanisms of cellulases and xylanases: a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi. Biochemistry. 1994;33(20):6363-70. DOI:10.1021/bi00186a041 |

3. pmid=IN Press

4. Andrews SR, Charnock SJ, Lakey JH, Davies GJ, Claeyssens M, Nerinckx W, Underwood M, Sinnott ML, Warren RA, and Gilbert HJ. (2000). 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;275(30):23027-33. DOI:10.1074/jbc.M000128200 |
5. Tull D, Withers SG, Gilkes NR, Kilburn DG, Warren RA, and Aebersold R. (1991). Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi. J Biol Chem. 1991;266(24):15621-5. | Google Books | Open Library
6. MacLeod AM, Lindhorst T, Withers SG, and Warren RA. (1994). The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants. Biochemistry. 1994;33(20):6371-6. DOI:10.1021/bi00186a042 |
7. Derewenda U, Swenson L, Green R, Wei Y, Morosoli R, Shareck F, Kluepfel D, and Derewenda ZS. (1994). 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;269(33):20811-4. | Google Books | Open Library
8. White A, Withers SG, Gilkes NR, and Rose DR. (1994). Crystal structure of the catalytic domain of the beta-1,4-glycanase cex from Cellulomonas fimi. Biochemistry. 1994;33(42):12546-52. DOI:10.1021/bi00208a003 |
9. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1995). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995;92(15):7090-4. DOI:10.1073/pnas.92.15.7090 |