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Glycoside Hydrolase Family 125

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


Substrate specificities

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]. Crystallographic evidence from a binary complex of CpGH125 with α-D-mannopyranosyl-(1–6)-α-D-mannopyranose, complemented by quantum mechanics/molecular mechanics calculations of preferred conformations on-enzyme and of reaction coordinate metadynamics, supports a 1S5B2,5OS2 conformational reaction coordinate [2].

Catalytic Residues

The structural analysis of CpGH125 suggested it uses aspartate 220 as a catalytic general acid and glutamate 393 as catalytic general 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 (thiomannobiose), 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 revealed the (α/α)6-fold of the family [1]. The complexes of SpGH125 with the inhibitor 1-deoxymannojirimycin and CpGH125 with the non-hydrolyzable substrate analogue methyl 1,6-α-thiomannobiose provided 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 provided a view of how more extensive substrates may be recognized by these enzymes. The uncomplexed structures of two GH125 enzymes from Bacteroides ovatus (3on6, and 3p2c) and one GH125 from Bacteroides thetaiotaomicron (2p0v) 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 and at present remain otherwise uncharacterized.

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 2p0v). 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. 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 Apr 29;286(17):15586-96. DOI:10.1074/jbc.M111.223172 | PubMed ID:21388958 | HubMed [Gregg2011]
  2. Alonso-Gil S, Males A, Fernandes PZ, Williams SJ, Davies GJ, and Rovira C. Computational Design of Experiment Unveils the Conformational Reaction Coordinate of GH125 α-Mannosidases. J Am Chem Soc. 2017 Jan 25;139(3):1085-1088. DOI:10.1021/jacs.6b11247 | PubMed ID:28026180 | HubMed [Alonso-Gil2017]
All Medline abstracts: PubMed | HubMed
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