Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

TY - JOUR

T1 - Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

AU - Larsbrink, Johan

AU - Izumi, Atsushi

AU - Hemsworth, Glyn R.

AU - Davies, Gideon J.

AU - Brumer, Harry

PY - 2012/12/21

Y1 - 2012/12/21

N2 - The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these alpha-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in alpha-glucan side chain (de) branching, regulation of oligo-and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-alpha-glucan 4-alpha-glucosyltransferase from GH31. Distinct from 1,4-alpha-glucan 6-alpha-glucosyltransferases (EC 2.4.1.24) and 4-alpha-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from alpha(1 -> 4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-beta-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

AB - The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these alpha-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in alpha-glucan side chain (de) branching, regulation of oligo-and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-alpha-glucan 4-alpha-glucosyltransferase from GH31. Distinct from 1,4-alpha-glucan 6-alpha-glucosyltransferases (EC 2.4.1.24) and 4-alpha-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from alpha(1 -> 4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-beta-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

KW - THERMUS-AQUATICUS

KW - ESCHERICHIA-COLI

KW - HUMAN MALTASE-GLUCOAMYLASE

KW - SUBSTRATE-SPECIFICITY

KW - THERMOTOGA-MARITIMA MALTOSYLTRANSFERASE

KW - CYCLIC GLUCANS

KW - STARCH METABOLISM

KW - ACTION PATTERN

KW - THERMOSTABLE 4-ALPHA-GLUCANOTRANSFERASE

KW - CRYSTAL-STRUCTURE

UR - http://www.scopus.com/inward/record.url?scp=84871601424&partnerID=8YFLogxK

U2 - 10.1074/jbc.M112.416511

DO - 10.1074/jbc.M112.416511

M3 - Article

SN - 0021-9258

VL - 287

SP - 43288

EP - 43299

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 52

ER -