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 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%.

The Characterization and Modification of a Novel Bifunctional and Robust Alginate Lyase Derived from Marinimicrobium sp. H1

“…As reported, AOS has been shown to be a bifidogenic factor that promotes plant growth and enhances the growth of human endothelial cells and keratinocytes [9]. AOS can be used in medicine and healthy food because it possesses special biological activities, such as anti-tumor, anti-inflammatory, anti-coagulation, anti-oxidation, immune regulation, and blood lipid and blood sugar lowering effects [10,11]. In addition, protoplast fragments produced during the degradation of alginate can be exploited as feed for animals [12].…”

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

“…However, there is growing interest in such simple but also in more complex enzyme cascades due to several advantages over whole-cell systems (Fessner, 2015). Such a cell-free enzyme cascade approach has been reported for alginate conversion to KDPG and further to GAP and pyruvate (Nishiyama et al, 2017). This is especially appealing for product formation from cheap and more easily available substrates and might also be considered as an alternative for KDPG synthesis.…”

Cobetia sp. Bacteria, Which Are Capable of Utilizing Alginate or Waste Laminaria sp. for Poly(3-Hydroxybutyrate) Synthesis, Isolated From a Marine Environment

“…These enzymes would constitute the metabolic pathway to convert alginate to DEH in the 5-11-6-3 and 5-25-4-2 strains ( Figure 4 ). Furthermore, the putative gene encoding 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes the cleavage of KDPG to glyceraldehyde-3-phosphate and pyruvate ( Nishiyama et al, 2017 ), was found in scaffold 1 (925,956 bp) of the 5-11-6-3 strain and in scaffold 1 (1,114,691 bp) of the 5-25-4-2 strain. However, the putative gene encoding 2-keto-3-deoxy-D-gluconate (KDG) kinase, which converts KDG to KDPG, was not found in the draft genome sequences of either strain.…”