Glycoside Hydrolase Family 140

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• Author: ^^^Alan Cartmell^^^
• Responsible Curator: ^^^Harry Gilbert^^^

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Glycoside Hydrolase Family GH140
Clan	GH-x
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH140.html

Substrate specificities

Thus far only one member of the family has been characterised, BT1012 from bacteroides thetaiotaomicron. BT1012 displays endo-apiosidase activity targeting α1,2 linked apiose at the base of the sidechains A and B in the complex glycan rhamnogalacturonan ii (RGII)[1]. Cleavage of the RGII backbone must occur for BT1012 to then act[1].

Kinetics and Mechanism

GH140 likely uses a, retaining, double displacement mechanism. This is strongly supported by methanolysis experiments where BT1012 was incubated with trisaccharide L-rhamnose-β1,3-D-apiose-α1,2-D-galacturonic acid-O-methyl in the presence of 10 % methanol. This experiment performed with BT1012 generated the product L-rhamnose-β1,3-D-apiose-O-methyl suggesting a retaining mechanism[1]. Glycoside hydrolases that utilise a retaining mechanism, but not those that use an inverting mechanism, can utilise methanol as an external nucleophile and thus generate a methylated product.

Catalytic Residues

The catalytic residues are an aspartate and glutamate located on the top of β-strands 4 and 7, respectively[1]. This could mean GH140 is a distant relative of Clan GH-A enzymes, however in with GH-A the catalytic residue atop of β-strands 4 and 7 are both glutamates. In the absence of a ligand bound complex or more detailed biochemical analysis [2] (preferably both) it is not possible to say which of the catalytic residues is the nucleophile or acid/base.

Three-dimensional structures

GH140 adopts a (β/α)₈ , TIM barrel, where a central barrel of eight β strands are encircled by eight α helices[1]. BT1012, the only GH140 structure available, also has a Ig like domain that stacks against the TIM barrel likely providing structural stability, similar to the role of Ig like domains in GH43 enzymes[3, 4].

Family Firsts

First stereochemistry determination

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Methanolysis experiments suggest a retaining mechanism[1].

First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

BT1012 from bacteroides thetaiotaomicron[1].

References

1. Ndeh D, Rogowski A, Cartmell A, Luis AS, Baslé A, Gray J, Venditto I, Briggs J, Zhang X, Labourel A, Terrapon N, Buffetto F, Nepogodiev S, Xiao Y, Field RA, Zhu Y, O'Neil MA, Urbanowicz BR, York WS, Davies GJ, Abbott DW, Ralet MC, Martens EC, Henrissat B, and Gilbert HJ. (2017). Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature. 2017;544(7648):65-70. DOI:10.1038/nature21725 |
2. Shallom D, Belakhov V, Solomon D, Shoham G, Baasov T, and Shoham Y. (2002). Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase. J Biol Chem. 2002;277(46):43667-73. DOI:10.1074/jbc.M208285200 |
3. McKee LS, Peña MJ, Rogowski A, Jackson A, Lewis RJ, York WS, Krogh KB, Viksø-Nielsen A, Skjøt M, Gilbert HJ, and Marles-Wright J. (2012). Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains. Proc Natl Acad Sci U S A. 2012;109(17):6537-42. DOI:10.1073/pnas.1117686109 |
4. Cartmell A, McKee LS, Peña MJ, Larsbrink J, Brumer H, Kaneko S, Ichinose H, Lewis RJ, Viksø-Nielsen A, Gilbert HJ, and Marles-Wright J. (2011). The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases. J Biol Chem. 2011;286(17):15483-95. DOI:10.1074/jbc.M110.215962 |
5. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |

6. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. Download PDF version.