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.

Recently, we identified an alginate-assimilating gene cluster in the genome of sp. strain UMI-01, a member of . 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., and , still remained uncharacterized. The amino acid sequences deduced from and showed low identities with those of corresponding enzymes of 2-40, a member of (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of species are somewhat deviated from those of species. 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 an expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several and species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%-25%) with the enzymes, while they showed relatively high identities (47%-68%) with the enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the enzymes and the enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes and 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%.