title: Changes in the catalytic properties of Pyrococcus furiosus thermostable amylase by mutagenesis of the substrate binding sites.
abstract: Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced alpha-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over beta-CD, calculated by comparing k(cat)/K(m) ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes.
authors.affiliation: Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151-921, Korea.
authors.forename: Sung-Jae
authors.initials: SJ
authors.lastname: Yang
authors.forename: Byoung-Chul
authors.initials: BC
authors.lastname: Min
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.forename: Sang-Mok
authors.initials: SM
authors.lastname: Jang
authors.forename: Byong-Hoon
authors.initials: BH
authors.lastname: Lee
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics*; D002384:Catalysis; D003505:Cyclodextrins / Q000378:metabolism*; D004795:Enzyme Stability; D005818:Genetic Engineering / Q000379:methods*; D006358:Hot Temperature; D007218:Industrial Microbiology / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D003505:Cyclodextrins; D000516:alpha-Amylases