title: Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.
abstract: Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, 17104, South Korea; Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup, 56212, South Korea.
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Jung
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, 17104, South Korea.
authors.forename: Min-Ji
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, 17104, South Korea.
authors.forename: Woo-Soo
authors.initials: WS
authors.lastname: Jeong
authors.affiliation: Korea Food Research Institute, Seongnam, 13539, South Korea.
authors.forename: Dong-Ho
authors.initials: DH
authors.lastname: Seo
authors.affiliation: Department of Bioengineering and Technology, Kangwon National University, Chuncheon, 24341, South Korea.
authors.forename: Suk-Jin
authors.initials: SJ
authors.lastname: Ha
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, 30019, South Korea.
authors.forename: Young Wan
authors.initials: YW
authors.lastname: Kim
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, 17104, South Korea. Electronic address: cspark@khu.ac.kr.
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
mesh_terms: D020816:Amino Acid Motifs; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology; D020134:Catalytic Domain; D017124:Conserved Sequence; D004187:Disaccharides / Q000378:metabolism; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D014644:Genetic Variation; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007534:Isomaltose / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D009834:Oligo-1,6-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D011994:Recombinant Proteins; D013395:Sucrose; D007534:Isomaltose; D005964:Glucosyltransferases; C116853:sucrose isomerase; D009834:Oligo-1,6-Glucosidase; C042003:trehalulose; C008189:isomaltulose
keywords: Azotobacter vinelandii; Oligo 1,6 glucosidase; RLDRD motif; Sucrose isomerase