Yasushi Tani

Eicosapentaenoic acid plays a beneficial role in membrane organization and cell division of a cold-adapted bacterium, Shewanella livingstonensis Ac10.

Shewanella livingstonensis Ac10, a psychrotrophic gram-negative bacterium isolated from Antarctic seawater, produces eicosapentaenoic acid (EPA) as a component of phospholipids at low temperatures. EPA constitutes about 5% of the total fatty acids of cells grown at 4 degrees C. We found that five genes, termed orf2, orf5, orf6, orf7, and orf8, are specifically required for the synthesis of EPA by targeted disruption of the respective genes. The mutants lacking EPA showed significant growth retardation at 4 degrees C but not at 18 degrees C. Supplementation of a synthetic phosphatidylethanolamine that contained EPA at the sn-2 position complemented the growth defect. The EPA-less mutant became filamentous, and multiple nucleoids were observed in a single cell at 4 degrees C, indicating that the mutant has a defect in cell division. Electron microscopy of the cells by high-pressure freezing and freeze-substitution revealed abnormal intracellular membranes in the EPA-less mutant at 4 degrees C. We also found that the amounts of several membrane proteins were affected by the depletion of EPA. While polyunsaturated fatty acids are often considered to increase the fluidity of the hydrophobic membrane core, diffusion of a small hydrophobic molecule, pyrene, in the cell membranes and large unilamellar vesicles prepared from the lipid extracts was very similar between the EPA-less mutant and the parental strain. These results suggest that EPA in S. livingstonensis Ac10 is not required for bulk bilayer fluidity but plays a beneficial role in membrane organization and cell division at low temperatures, possibly through specific interaction between EPA and proteins involved in these cellular processes.

Novel Neogala-Series Glycosphingolipids with a Terminal Glucose Residue from the Fungus Mariannaea elegans

Glycosphingolipids (GSLs) are essential membrane components of eukaryotic cells. Recently, a new type of fungal neogala-series GSL was identified in aureobasidin A-resistant fungi. In this study, we analyzed GSLs from four pathogenic fungal strains belonging to the order Hypocreales, and found that Mariannaea elegans contained both acidic GSLs and neutral GSLs with mono- and di-saccharides. The structures of the neutral GSLs of M. elegans were determined by compositional sugar, fatty acid, and sphingoid analyses by GC/MS, MALDI time-of-flight/MS, and 1H NMR. The ceramide moiety of Glcβ1-Cer consisted mainly of the 2-hydroxylated C18:0-fatty acid 9-methyl-octadeca-4-sphinganine or 9-methyl-octadeca-4,8-sphingadienine. In contrast, the ceramides of Galβ1-6Galβ1-Cer and Glc1-6Galβ1-Cer consisted mainly of saturated 2-hydroxylated C24:0-fatty acids and C18:0-phytosphingosine. To our knowledge, Glc1-6Galβ1-Cer is a novel GSL in fungi, and M. elegans is the first example of an aureobasidin A-sensitive fungus that possesses fungal neogala series GSLs.

Purification and properties of 4-methyl-5-hydroxyethylthiazole kinase from Escherichia coli.

4-Methyl-5-hydroxyethylthiazole kinase (ThiM) participates in thiamin biosynthesis as the key enzyme in its salvage pathway. We purified and characterized ThiM from Escherichia coli. It has broad substrate specificity toward various nucleotides and shows a preference for dATP as a phosphate donor over ATP. It is activated by divalent cations, and responds more strongly to Co2+ than to Mg2+.

Heterologous expression of l-lysine α-oxidase from Scomber japonicus in Pichia pastoris and functional characterization of the recombinant enzyme

Fish have a complex self-defense mechanism against microbial invasion. Recently, l-lysine α-oxidases have been identified from a number of fish species as a novel type of antibacterial protein in the integument. These enzymes exhibit strict substrate specificity for l-lysine, but the underlying mechanisms and details of their catalytic properties remain unknown. In this study, a synthetic gene coding for Scomber japonicus l-lysine α-oxidase, originally termed AIP (for apoptosis-inducing protein), was expressed in Pichia pastoris, and the recombinant enzyme (rAIP) was purified and characterized. rAIP exhibited essentially the same substrate specificity as the native enzyme, catalyzing the oxidative deamination of l-lysine as an exclusive substrate. rAIP was N-glycosylated and remained active over a wide range of pH, with an optimal pH of 7.5. The enzyme was stable in the pH range from 4.5 to 10.0 and was thermally stable up to 60°C. A molecular modelling of rAIP and a comparative structure/sequence analysis with homologous enzymes indicate that Asp(220) and Asp(320) are the substrate-binding residues that are likely to confer exclusive substrate specificity for l-lysine on the fish enzymes.

Purification and Properties of Glycine Oxidase from Pseudomonas putida KT2440

Glycine oxidase, encoded by the thiO gene, participates in the biosynthesis of thiamin by providing glyoxyl imine to form the thiazole moiety of thiamin. We have purified and characterized ThiO from Pseudomonas putida KT2440. It has a monomeric structure that is distinct from the homotetrameric ThiOs from Bacillus subtilis and Geobacillus kaustophilus. The P. putida ThiO is unique in that glycine is its preferred substrate, which differs markedly from the B. subtilis and G. kaustophilus enzymes that use D-proline as the preferred substrate.