Glycoside Hydrolase Family 145

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

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Glycoside Hydrolase Family GH145
Clan	GH-x
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH145.html

Substrate specificities

Two members of this family have been shown to be α-L-rhamnosidases, targeting rhamnose linked α-1,4 to glucuronic acid in the complex arabinogalactan protein gum arabic [1, 2].

Kinetics and Mechanism

NMR, using the arabinogalactan protein (AGP) gum arabic as the substrate, revealed the family operates via a retaining mechanism[1]. Rather than using a standard double displacement mechanism the enzyme is speculatively predicted to perform catalysis via an epoxide intermediate, similar to GH99 enzymes[3, 4, 5]. GH145, however, is proposed to perform catalysis via a substrate assisted mechanism, requiring the carboxyl group of the glucuronic acid and a single catalytic histidine; both acting as an acid/base. This histidine is predicted to deprotonate the O2 of rhamnose, allowing O2 to attack C1 and form an epoxide. Simultaneously the carboxyl group of the glucuronic acid may deprotonate a water molecule generating a hydroxyl group to attach the C1 of rhamnose and allowing protonation of its own O4 thus, leading to glycosidic bond cleavage[1]. Further work is needed to confirm the mechanism by which GH145 operates.

Catalytic Residues

A single catalytic histidine has been shown to be critical for activity. The introduction of the catalytic histidine into related enzymes, which lack the histidine and rhamnosidase activity, is sufficient to introduce rhamnosidase activity into these enzymes[1].

Three-dimensional structures

GH145 comprise a single domain which is a seven bladed β-propeller fold. Each blade is composed of four anti parallel β-strands that extend out radially from the central core. The final blade is formed by strands from both the N- and C-terminus of the protein which is termed as 'molecular velcro' and is believed to add considerable stability to the fold. The active site of these α-L-rhamnosidases is located on the opposite side, termed the posterior surface, of CAZymes with similar β-propeller folds. The "normal" side, termed the anterior surface, of the β-propeller bears the highest residue conservation and may well have another function. GH145 is distantly related to PL25 which utilise the anterior surface suggesting that the this surface in GH145 may have another activity[1, 6].

Family Firsts

First stereochemistry determination

Determined for the bacteroides thetaiotaomicron enzyme BT3686

First catalytic acid/base residue identification

Predicted to be a histidine

Second general acid/base residue identification

Predicted to be provided by the substrate.

First 3-D structure

BT3686, BACINT_00347 and BACCELL_00856 were the first enzymes to have their structures solved.

References

<biblio>

1. Munoz-Munoz2017 pmid=28396425
2. Cartmell2019 pmid=31541200
3. Thompson2012 pmid=22219371
4. Fernandes2018 pmid=29508463
5. Ulaganathan2017 pmid=28290654
6. Cantarel2009 pmid=18838391
7. DaviesSinnott2008 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.