title: Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01.
abstract: Recently, we identified an alginate-assimilating gene cluster in the genome ofFlavobacteriumsp. strain UMI-01, a member ofBacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e.,flkinandflald, still remained uncharacterized. The amino acid sequences deduced fromflkinandflaldshowed low identities with those of corresponding enzymes ofSaccharophagus degradans2-40T, a member ofProteobacteria(Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes ofBacteroidetesspecies are somewhat deviated from those ofProteobacteriaspecies. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by anEscherichia coliexpression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from severalProteobacteriaandBacteroidetesspecies indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%-25%) with theProteobacteriaenzymes, while they showed relatively high identities (47%-68%) with theBacteroidetesenzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between theProteobacteriaenzymes and theBacteroidetesenzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genesflkinandflald,respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium state. An in vitro alginate-metabolizing system constructed from recFlKin, recFlAld, and previously reported alginate lyases and DEH reductase of the strain UMI-01 could convert alginate to pyruvate and glyceraldehyde-3-phosphate with an efficiency of 38%.
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. nsym2480rj@eis.hokudai.ac.jp.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Nishiyama
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. inouea21@fish.hokudai.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Inoue
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. ojima@fish.hokudai.ac.jp.
authors.forename: Takao
authors.initials: T
authors.lastname: Ojima
mesh_terms: D000446:Aldehyde-Lyases / Q000378:metabolism*; D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D041963:Bacteroidetes / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005417:Flavobacterium / Q000378:metabolism*; D005942:Gluconates / Q000378:metabolism*; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010802:Phylogeny; D020560:Proteobacteria / Q000378:metabolism; D016415:Sequence Alignment; D014574:Uronic Acids / Q000378:metabolism
chemical_list: D000464:Alginates; D005942:Gluconates; D006603:Hexuronic Acids; D014574:Uronic Acids; C081217:2-keto-3-deoxy-6-phosphogluconate; D020723:Glucuronic Acid; D010088:Oxidoreductases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021471:2-keto-3-deoxygluconokinase; D000446:Aldehyde-Lyases; C100131:phospho-2-keto-3-deoxy-gluconate aldolase
keywords: ">d-gluconate (KDG) kinase; ">l-erythro-5-hexoseulose uronic acid (DEH) metabolism; 2-keto-3-deoxy-; 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase; 4-deoxy-; Bacteroidetes; Flavobacterium; Proteobacteria; alginate degradation; alginate-derived products