Title
Identification of acceptor substrate binding subsites +2 +3 in the amylomaltase from Thermus thermophilus HB8
Author
Kaper, T.
Leemhuis, H.
Uitdehaag, J.C.M.
van der Veen, B.A.
Dijkstra, B.W.
van der Maarel, M.J.E.C.
Dijkhuizen, L.
TNO Kwaliteit van Leven
Publication year
2007
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
Subject
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
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http://resolver.tudelft.nl/uuid:7f6431fe-e966-467a-b438-f6a3595ee5b8
DOI
https://doi.org/10.1021/bi602408j
TNO identifier
43706
Source
Biochemistry, 46 (17), 5261-5269
Document type
article