Glycoside Hydrolase Family 125

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• Author: ^^^Alisdair Boraston^^^
• Responsible Curator: ^^^Alisdair Boraston^^^

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Glycoside Hydrolase Family GH125
Clan	GH-L
Mechanism	inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/GH125.html

Substrate specificities

The currently characterized family 125 glycoside hydrolases, which include the examples from Streptococcus pneumoniae (SpGH125) and Clostridium perfringens (CpGH125), are α-mannosidases with specificity for α-1,6-linked non-reducing terminal mannose residues [1].

Kinetics and Mechanism

Kinetic characterization of 2,4-dinitrophenyl α-D-mannopyranoside hydrolysis by SpGH125 and CpGH125 revealed that this is a relatively poor substrate for these enzymes. Monitoring the hydrolysis of methyl 6-O-(α-D-mannopyranosyl)-β-D-mannopyranoside by ¹H NMR spectroscopy showed that CpGH125 and SpGH125 act with inversion of stereochemistry. The structural analysis of both enzymes detailed an arrangement of catalytic residues that was consistent with this mechanistic assignment [1].

Catalytic Residues

The structural analysis of CpGH125 suggests it uses aspartate 220 as a catalytic acid and glutamate 393 as catalytic base. The corresponding residues in SpGH125 are aspartate 218 and glutamate 391 [1]. The assignment of these residues was determined primarily from the structure of CpGH125 in complex with the non-hydrolyzable substrate-analog methyl-S-(α-D-mannopyranosyl)-(1–6)-α-D-mannopyranose, which spanned the -1 and +1 subsites and engaged the catalytic machinery. Additional support for the assignment was provided by structural alignment with other members of Clan GH-L, specifically GH15 and GH65, which revealed conservation of these catalytic residues among all of the clan members.

Three-dimensional structures

The three dimensional structures of CpGH125 (3qt3, 3qt9, and 2nvp), SpGH125 (3qpf, 3qry, and 3qsp) have been determined by X-ray crystallography and reveal the (α/α)₆-fold of the family. The complexes of SpGH125 with the inhibitor 1-deoxymannojirimycin and CpGH125 with the non-hydrolyzable substrate-analog methyl-S-(α-D-mannopyranosyl)-(1–6)-α-D-mannopyranose (thiomannobiose), the latter of which spans the -1 and +1 sub sites and engages the catalytic machinery, provide insight into the mode of substrate recognition, the identity of the catalytic residues, and the catalytic mechanism. A non-productive complex of SpGH125 with α-D-mannopyranosyl-(1–6)-α-D-mannopyranose occupying the +1 and +2 subsites provides a view of how more extensive substrates may be recognized by these enzymes. The uncomplexed structures two GH125 enzymes from Bacteroides ovatus (3on6, and 3p2c) and one GH125 from Bacteroides thetaiotaomicron (2pov) are notable as they were the first deposited structures for members of GH family 125; however, they were deposited prior to the determination of an activity for this family of enzymes.

Family Firsts

First stereochemistry determination

¹H NMR spectroscopy revealed that CpGH125 and SpGH125 act with inversion of stereochemistry [1].

First general base identification

CpGH125 and SpGH125 (inferred from structure) [1].

First general acid identification

CpGH125 and SpGH125 (inferred from structure) [1].

First 3-D structure

The first deposited structure was that of CpGH125 (2nvp) closely followed by the deposition of structures of the Bacteroides sp. proteins (3on6, 3p2c, and 2pov). These structures were determined by the Structural Genomics Consortium but not published. The first published structures were those of CpGH125 and SpGH125, which also presented the first structures of these proteins in complex with carbohydrates [1].

References

1. Gregg KJ, Zandberg WF, Hehemann JH, Whitworth GE, Deng L, Vocadlo DJ, and Boraston AB. (2011). Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans. J Biol Chem. 2011;286(17):15586-96. DOI:10.1074/jbc.M111.223172 |