Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149

Abstract

Two distinct bacterial and eukaryotic serine racemases (SRs) have been identified based on phylogenetic and biochemical characteristics. Although some reports have suggested that marine heterotrophic bacteria have the potential to produce D-serine, the gene encoding bacterial SRs is not found in those bacterial genomes. In this study, using in-depth genomic analysis, we found that eukaryotic SR homologues were distributed widely in various bacterial genomes. Additionally, we selected a eukaryotic SR homologue from a marine heterotrophic bacterium, Roseobacter litoralis Och 149 (RiSR), and constructed an RiSR gene expression system in Escherichia coli for studying the properties of the enzyme. Among the tested amino acids, the recombinant RiSR exhibited both racemization and dehydration activities only towards serine, similar to many eukaryotic SRs. Mg 2+ and MgATP enhanced both activities of RiSR, whereas EDTA abolished these enzymatic activities. The enzymatic properties and domain structure of RiSR were similar to those of eukaryotic SRs, particularly mammalian SRs. However, RiSR showed lower catalytic efficiency for L-serine dehydration (k cat /K m 50.094 min 21 mM 21 ) than those of eukaryotic SRs reported to date (k cat /K m 50.6-21 min 21 mM 21 ). In contrast, the catalytic efficiency for L-serine racemization of RiSR (k cat /K m 53.14 min 21 mM 21 ) was 34-fold higher than that of L-serine dehydration. These data suggested that RiSR primarily catalysed serine racemization rather than dehydration.

Enantioselective Utilization of D-Amino Acids by Deep-Sea Microorganisms

“…Microbiological and geochemical studies suggested the microbial production and consumption of DAAs in pelagic water ecosystems (Perez et al, 2003 ; Teira et al, 2006 ; Calleja et al, 2013 ; Zhang et al, 2016 ). In this geochemical cycle, importance of marine Alphaproteobacteria in the production of DAAs has already been pointed out (Pedersen et al, 2001 ; Lomstein et al, 2006 ; Kaiser and Benner, 2008 ; Kubota et al, 2016 ), however, phylogenetic and physiological features of DAA-utilizing marine bacteria are poorly investigated yet.…”

Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

“…R F value of the separated spots was calculated by using L-serine as a reference compound and the given formula: RP-HPLC-For the HPLC analysis, reverse-phase C-18 column (Agilent, C-18, X 4.6mm, 5um) and Agilent (1220 In nity II LC) HPLC system. Gradient elution was applied as described elsewhere with further modi cations [25]. The solvent system was set as: A. sodium acetate (pH 6.5, 40mM); B. methanol: acetonitrile (30:70).…”

Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

“…RP-HPLC-For the HPLC analysis, reverse-phase C-18 column (Agilent, C-18, 250 X 4.6mm, 5um) and Agilent (1220 In nity II LC) HPLC system. Gradient elution was applied as described elsewhere with further modi cations [25]. The solvent system was set as: A. sodium acetate (pH 6.5, 40mM); B.…”

“…Howbeit, our knowledge in the case of prokaryotic SRs is still constrained. For instance, the synthesis of D-serine in marine heterotrophs is unstable and inhibits the bacterial growth of Roseobacter litoralis Och 149 [25]. In Enterococcus gallinarum BM4174, Alanine racemase is known to synthesize D-serine which is the further precursor of PG of this Gram-positive bacteria [13].…”