Identification of Acceptor Substrate Binding Subsites +2 and +3 in the Amylomaltase from Thermus thermophilus HB8
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- 作者：Thijs Kaper ; Hans Leemhuis ; Joost C. M. Uitdehaag ; Bart A. van der Veen ; Bauke W. Dijkstra ; Marc J. E. C. van der Maarel ; Lubbert Dijkhuizen
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 1, 2007
- 年：2007
- 卷：46
- 期：17
- 页码：5261 - 5269
- 全文大小：160K
- 年卷期：v.46,no.17(May 1, 2007)
- ISSN：1520-4995
文摘
Glycoside hydrolase family 77 (GH77) belongs to the 
-amylase superfamily (Clan H) togetherwith GH13 and GH70. GH77 enzymes are amylomaltases or 4--glucanotransferases, involved in maltosemetabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltasefrom the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesisof the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transitionstate 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 isamong the most efficient 4--glucanotransferases in the -amylase superfamily. The active site containsat 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 theinteractions 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 G7as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of -amylases only upto 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13amylases and GH77 amylomaltases.
-amylase superfamily (Clan H) togetherwith GH13 and GH70. GH77 enzymes are amylomaltases or 4--glucanotransferases, involved in maltosemetabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltasefrom the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesisof the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transitionstate 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 isamong the most efficient 4--glucanotransferases in the -amylase superfamily. The active site containsat 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 theinteractions 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 G7as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of -amylases only upto 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13amylases and GH77 amylomaltases.