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

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3. pmid=IN Press

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 |