**Ryuji Nishiyama, Akira Inoue and Takao Ojima \***

Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan; nsym2480rj@eis.hokudai.ac.jp (R.N.); inouea21@fish.hokudai.ac.jp (A.I.) **\*** Correspondence: ojima@fish.hokudai.ac.jp; Tel./Fax: +81-138-40-8800

Academic Editor: Antonio Trincone

Received: 28 October 2016; Accepted: 8 February 2017; Published: 14 February 2017

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

**Keywords:** alginate degradation; 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) metabolism; *Bacteroidetes*; *Proteobacteria*; *Flavobacterium*; 2-keto-3-deoxy-D-gluconate (KDG) kinase; 2-keto-3 deoxy-6-phosphogluconate (KDPG) aldolase; alginate-derived products
