Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8.
Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-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-alpha-glucanotransferases in the alpha-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 GH13 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 alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.
Kaper
Thijs
T
Centre for Carbohydrate Bioprocessing TNO, University of Groningen, 9751 NN Haren, The Netherlands.
Leemhuis
Hans
H
Uitdehaag
Joost C M
JC
van der Veen
Bart A
BA
Dijkstra
Bauke W
BW
van der Maarel
Marc J E C
MJ
Dijkhuizen
Lubbert
L
D020909:Acarbose / Q000494:pharmacology; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004795:Enzyme Stability; D006004:Glycogen Debranching Enzyme System / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
D006004:Glycogen Debranching Enzyme System; C020569:4 alpha-glucanotransferase; D020909:Acarbose