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Glycoside Hydrolase Family 31
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|Glycoside Hydrolase Family GH31|
|Active site residues||known|
|CAZy DB link|
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.  GH31 enzymes (except for the α-glucan lyases) are believed to follow the classical Koshland double-displacement mechanism.  This has been strongly supported by labeling of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide , with early examples including rabbit intestinal sucrase/isomaltase  and human lysosomal α-glucosidase . Later studies on an α-glucosidase from Aspergillus niger , an α-xylosidase from Escherichia coli , and an α-xylosidase from Cellvibrio japonicus  used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates. Subsequently, retention of the anomeric configuration was directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by C. japonicus Xyl31A 
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.
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 .
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 . This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from Escherichia coli  and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 .
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 . 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 .
The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from Escherichia coli, published in 2005 . Since that time, a number of structures have continued to emerge. Among these, the crystallographic study of the Sulfolobus solfataricus α-glucosidase (MalA) is notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of GH27 and GH36 ; these three families now compose clan GH-D. The structure of the N-terminal domain of human intestinal maltase-glucoamylase was the first from a eukaryotic member of GH31 . These structures reveal a common (β/α)8 barrel catalytic domain. Most GH31 members are multi-domain proteins, while the specific function (if any) of these accessory domains is generally unknown. An exception is the α-xylosidase, CjXyl31A of C. japonicus, in which a PA-14 domain that is rare among GH31 members is suggested to confer increased catalytic specificity toward large oligosaccharide substrates [8, 9].
- First stereochemical outcome
- Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements 
- First catalytic nucleophile identification
- Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling 
- First general acid/base residue identification
- Schizosaccharomyces pombe α-glucosidase by sequence comparison and kinetic studies of the mutants 
- First three-dimensional structure of GH31 enzymes
- Escherichia coli α-xylosidase (YicI) 
- Chiba S, Hiromi K, Minamiura N, Ohnishi M, Shimomura T, Suga K, Suganuma T, Tanaka A, Tomioka S, and Yamamoto T. Quantitative study on anomeric forms of glucose produced by alpha-glucosidases. J Biochem. 1979 May;85(5):1135-41.
- Frandsen TP and Svensson B. Plant alpha-glucosidases of the glycoside hydrolase family 31. Molecular properties, substrate specificity, reaction mechanism, and comparison with family members of different origin. Plant Mol Biol. 1998 May;37(1):1-13.
- Iwanami S, Matsui H, Kimura A, Ito H, Mori H, Honma M, and Chiba S. Chemical modification and amino acid sequence of active site in sugar beet alpha-glucosidase. Biosci Biotechnol Biochem. 1995 Mar;59(3):459-63.
- Quaroni A and Semenza G. Partial amino acid sequences around the essential carboxylate in the active sites of the intestinal sucrase-isomaltase complex. J Biol Chem. 1976 Jun 10;251(11):3250-3.
- Hermans MM, Kroos MA, van Beeumen J, Oostra BA, and Reuser AJ. Human lysosomal alpha-glucosidase. Characterization of the catalytic site. J Biol Chem. 1991 Jul 25;266(21):13507-12.
- Lee SS, He S, and Withers SG. Identification of the catalytic nucleophile of the Family 31 alpha-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate. Biochem J. 2001 Oct 15;359(Pt 2):381-6.
- Lovering AL, Lee SS, Kim YW, Withers SG, and Strynadka NC. Mechanistic and structural analysis of a family 31 alpha-glycosidase and its glycosyl-enzyme intermediate. J Biol Chem. 2005 Jan 21;280(3):2105-15. DOI:10.1074/jbc.M410468200 |
- Larsbrink J, Izumi A, Ibatullin FM, Nakhai A, Gilbert HJ, Davies GJ, and Brumer H. Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification. Biochem J. 2011 Jun 15;436(3):567-80. DOI:10.1042/BJ20110299 |
- Silipo A, Larsbrink J, Marchetti R, Lanzetta R, Brumer H, and Molinaro A. NMR spectroscopic analysis reveals extensive binding interactions of complex xyloglucan oligosaccharides with the Cellvibrio japonicus glycoside hydrolase family 31 α-xylosidase. Chemistry. 2012 Oct 15;18(42):13395-404. DOI:10.1002/chem.201200488 |
- Lee SS, Yu S, and Withers SG. alpha-1,4-Glucan lyase performs a trans-elimination via a nucleophilic displacement followed by a syn-elimination. J Am Chem Soc. 2002 May 8;124(18):4948-9.
- Lee SS, Yu S, and Withers SG. Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase. Biochemistry. 2003 Nov 11;42(44):13081-90. DOI:10.1021/bi035189g |
- Okuyama M, Okuno A, Shimizu N, Mori H, Kimura A, and Chiba S. Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe. Eur J Biochem. 2001 Apr;268(8):2270-80.
- Kim YW, Lovering AL, Chen H, Kantner T, McIntosh LP, Strynadka NC, and Withers SG. 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 Feb 22;128(7):2202-3. DOI:10.1021/ja057904a |
- Ernst HA, Lo Leggio L, Willemoës M, Leonard G, Blum P, and Larsen S. Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31. J Mol Biol. 2006 May 12;358(4):1106-24. DOI:10.1016/j.jmb.2006.02.056 |
- Sim L, Quezada-Calvillo R, Sterchi EE, Nichols BL, and Rose DR. Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. J Mol Biol. 2008 Jan 18;375(3):782-92. DOI:10.1016/j.jmb.2007.10.069 |