Glycoside Hydrolase Family 31

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• Author: ^^^Ran Zhang^^^
• Responsible Curator: Steve Withers

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Glycoside Hydrolase Family GH31
Clan	GH-D
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH31.html

Substrate specificities

CAZy Family GH31 is one of the two major families of glycoside hydrolases, along with GH13, that contain α-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal α-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to α-glucosidases, GH31 also contains α-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic α-glucan lyases. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.

Kinetics and Mechanism

Family GH 31 enzymes are retaining α-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. [1] GH31 enzymes (except for the α-glucan lyases) are believed to follow the classical Koshland double-displacement mechanism. [2] This has been strongly supported by labeling of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide [3], with early examples including rabbit intestinal sucrase/isomaltase [4] and human lysosomal α-glucosidase [5]. Later studies on an α-glucosidase from Aspergillus niger [6], an α-xylosidase from Escherichia coli [7], and an α-xylosidase from Cellvibrio japonicus [8] used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates. Retention of the anomeric configuration has been directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by C. japonicus Xyl31A [9]

The α-glucan lyases from GH31 cleave α-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see Lexicon). Detailed mechanistic studies have been carried out on Gracilariopsis α-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step [10, 11]. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-β-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two α-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-α-D-glucopyranosyl fluoride.

Catalytic Residues

Measurements of pH profiles suggested that two essential residues were involved in catalysis [2, 7, 11]. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of Aspergillus niger α-glucosidase within the sequence IDM [3, 5]. This was confirmed by using the more reliable 5-fluoro-α-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS [6].

The general acid/base residue was first tentatively assigned as Asp647 in the Schizosaccharomyces pombe α-glucosidase based on sequence comparison and kinetic analysis of the mutants [12]. This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from Escherichia coli [7] and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 [13].

The catalytic nucleophile in Gracilariopsis α-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-β-L-idopyranosyl fluoride [10]. This corresponds precisely to the position of the catalytic nucleophile in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs [11].

Three-dimensional structures

The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from Escherichia coli , published in 2005 [7]. To date two other crystal structures of GH31 enzymes have been published, one being the Sulfolobus solfataricus α-glucosidase (MalA) [14] and the other being the N-terminal domain of human intestinal maltase-glucoamylase [15]. All of these structures feature a (β/α)₈ barrel in the catalytic domain. The Sulfolobus solfataricus α-glucosidase (MalA) study is especially notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of GH27 and GH36 [14]. These three families now compose clan GH-D.

Family Firsts

First stereochemical outcome

Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements [1]

First catalytic nucleophile identification

Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling [4]

First general acid/base residue identification

Schizosaccharomyces pombe α-glucosidase by sequence comparison and kinetic studies of the mutants [12]

First three-dimensional structure of GH31 enzymes

Escherichia coli α-xylosidase (YicI) [7]

References

1. Chiba S, Hiromi K, Minamiura N, Ohnishi M, Shimomura T, Suga K, Suganuma T, Tanaka A, Tomioka S, and Yamamoto T. (1979). Quantitative study on anomeric forms of glucose produced by alpha-glucosidases. J Biochem. 1979;85(5):1135-41. | Google Books | Open Library
8. Larsbrink J, Izumi A, Ibatullin FM, Nakhai A, Gilbert HJ, Davies GJ, and Brumer H. (2011). Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification. Biochem J. 2011;436(3):567-80. DOI:10.1042/BJ20110299 |
9. Silipo A, Larsbrink J, Marchetti R, Lanzetta R, Brumer H, and Molinaro A. (2012). NMR spectroscopic analysis reveals extensive binding interactions of complex xyloglucan oligosaccharides with the Cellvibrio japonicus glycoside hydrolase family 31 α-xylosidase. Chemistry. 2012;18(42):13395-404. DOI:10.1002/chem.201200488 |
10. Lee SS, Yu S, and Withers SG. (2002). alpha-1,4-Glucan lyase performs a trans-elimination via a nucleophilic displacement followed by a syn-elimination. J Am Chem Soc. 2002;124(18):4948-9. DOI:10.1021/ja0255610 |
11. Lee SS, Yu S, and Withers SG. (2003). Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase. Biochemistry. 2003;42(44):13081-90. DOI:10.1021/bi035189g |
12. Okuyama M, Okuno A, Shimizu N, Mori H, Kimura A, and Chiba S. (2001). Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe. Eur J Biochem. 2001;268(8):2270-80. DOI:10.1046/j.1432-1327.2001.02104.x |
13. Kim YW, Lovering AL, Chen H, Kantner T, McIntosh LP, Strynadka NC, and Withers SG. (2006). Expanding the thioglycoligase strategy to the synthesis of alpha-linked thioglycosides allows structural investigation of the parent enzyme/substrate complex. J Am Chem Soc. 2006;128(7):2202-3. DOI:10.1021/ja057904a |
14. Ernst HA, Lo Leggio L, Willemoës M, Leonard G, Blum P, and Larsen S. (2006). Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31. J Mol Biol. 2006;358(4):1106-24. DOI:10.1016/j.jmb.2006.02.056 |
15. Sim L, Quezada-Calvillo R, Sterchi EE, Nichols BL, and Rose DR. (2008). Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. J Mol Biol. 2008;375(3):782-92. DOI:10.1016/j.jmb.2007.10.069 |