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Identification of acceptor substrate binding subsites +2 +3 in the amylomaltase from Thermus thermophilus HB8

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Author: Kaper, T. · Leemhuis, H. · Uitdehaag, J.C.M. · Veen, B.A. van der · Dijkstra, B.W. · Maarel, M.J.E.C. van der · Dijkhuizen, L.
Type:article
Date:2007
Institution: TNO Kwaliteit van Leven
Source:Biochemistry, 17, 46, 5261-5269
Identifier: 43706
doi: doi:10.1021/bi602408j
Keywords: Nutrition · Food technology · Acarbose · Amino Acid Sequence · Catalytic Domain · Enzyme Stability · Glycogen Debranching Enzyme System · Hydrogen-Ion Concentration · Hydrolysis · Kinetics · Molecular Sequence Data · Substrate Specificity · Thermus thermophilus · Bacteria (microorganisms) · Catalysts · Enzymes · Mutagenesis · Oligosaccharides · Amylomaltase · Glucanotransferases · Glycoside hydrolase · Substrates

Abstract

Glycoside hydrolase family 77 (GH77) belongs to the α-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-α-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-α-glucanotransferases in the α-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GHl3 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of a-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases. © 2007 American Chemical Society. Chemicals / CAS: 4alpha glucanotransferase, 9032-09-1; amylase, 9000-90-2, 9000-92-4, 9001-19-8; glycosidase, 9032-92-2; 4 alpha-glucanotransferase, EC 2.4.1.25; Acarbose, 56180-94-0; Glycogen Debranching Enzyme System