Akio Suemori

Pseudomonas alcaliphila sp. nov., a novel facultatively psychrophilic alkaliphile isolated from seawater

Facultatively psychrophilic alkaliphilic strains were isolated from seawater obtained off the coast of Rumoi, Hokkaido, Japan. They were Gram-negative, aerobic straight rods with polar flagella. The isolates were catalase- and oxidase-positive and able to grow at 4 degrees C, but not at 40 degrees C. They produced acid from D-glucose under aerobic conditions. The isolates reduced nitrate to nitrite and hydrolysed casein and gelatin, but not starch or DNA. NaCl was required for growth at pH 10 but was not required at neutral pH. The major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 62.3-63.2 mol%. The whole-cell fatty acids mainly consisted of C16:0, C16:1(9c) and C18:1(9c), with 3-OH C10:0 and 3-OH C12:0 as the hydroxyl fatty acids. A larger amount of trans-unsaturated fatty acid, C16:1(9t) was observed when the cells were grown at pH 7 compared to when cells were grown at pH 10. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacteria are members of the genus Pseudomonas. Analysis of DNA-DNA relatedness data with several close phylogenetic neighbours revealed a low level of hybridization (less than 61%). On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, it is concluded that these isolates represent a separate new species. Accordingly, the name Pseudomonas alcaliphila is proposed. The type strain is AL15-21T (= JCM 10630T = IAM 14884T).

A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

Conserved and non-conserved residues and their role in the structure and function of p-hydroxybenzoate hydroxylase

In order to elucidate the molecular mechanism of the catalytic reaction and enzyme conformation, we substituted 53 conserved residues identified by aligning 92 p-hydroxybenzoate hydroxylase sequences and 19 non-conserved residues selected from crystallographic studies of Pseudomonas fluorescens NBRC14160 p-hydroxybenzoate hydroxylase with 19 other naturally occurring amino acids, yielding a database of 619 active single mutants. The database contained 365 and 254 active single mutants for 44/53 conserved residues and 19 non-conserved residues, respectively; the data included main activity, sub-activity for NADPH and NADPH reaction specificity. Active mutations were not observed for the G14, Q102, G160, E198, R220, R246, N300, F342 and G387 conserved residues, and only one active mutant was obtained at the G9, G11, G187, D286, Y201, R214 and G295 conserved residues and the S13, E32 and R42 non-conserved residues. Only seven active mutants with higher activity than the wild-type enzyme were observed at conserved residues, and only two were observed at non-conserved residues. The 365 mutants at conserved residues included 64 active mutants with higher NADPH reaction specificity than the wild-type enzyme, and some Y181X single mutants exhibited considerable changes in NADPH reaction specificity. A Y181X/L268G double-mutant database was constructed to computationally analyze the effects of these substitutions on structural conformation and function. These results indicated that some conserved or non-conserved residues are important for structural stability or enzyme function.

Location of flavoprotein-aromatic compound oxygenases from Rhodococcus erythropolis

Abstract Gram-positive Rhodococcus erythropolis strain S-1, grown in an aromatic carboxylic acid-salts medium, synthesized enzymes for the degradation of aromatic carboxylic acids, phthalate, monohydroxybenzoates, and dihydroxybenzoates. Flavoprotein-aromatic compound monooxygenases and dioxygenase from R. erythropolis, involved in the initial steps of the degradation pathways of aromatic carboxylic acids, were found in the membrane fraction. The enzymes in the membrane fraction were solubilized by Brij 58 or Tween 80 but denatured by Triton X-100. Solubilized and purified enzyme stability was conserved by Tween 80.

Temperature- and detergent-dependent oligomeric structures of flavoprotein monohydroxybenzoate hydroxylases from Rhodococcus erythropolis

The results of analytical gel filtration chromatography experiments under various conditions showed each of three flavoprotein monohydroxybenzoate hydroxylases, p-hydroxybenzoate 3-hydroxylase, m-hydroxybenzoate 6-hydroxylase, and salicylate 5-hydroxylase, from the gram-positive Rhodococcus erythropolis strain S1 to be present in solution mainly as a tetramer at 4°C and as a dimer at 30°C. In the presence of 1% Tween 80, the three flavoenzymes were each observed to be a monomer with activity about half that of the dimer.

Engineering of histidine tail in the N-terminal region of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The pobA of Pseudomonas florescens IFO14160 encoding a p-hydroxybenzoate hydroxylase (PHBH) was cloned, sequenced, and over-expressed in Escherichia coli. To facilitate the purification of PHBH, a fully active, tagged enzyme was constructed by engineering a two-, three-, or six-histidine tail in the N-terminal region (H2-, H3-, or H6-PHBH), or a six-histidine tail in the C-terminal region of the PHBH. The six-histidine tail in the C-terminal region of the enzyme could not be expressed with activity, while the other polyhistidine tails in the N-terminal region of the enzyme were highly active. However, the resulting H6-PHBH could not be purified by Ni-NTA chromatography because the H6-PHBH was so strongly bound to the supports that it could be eluted only after a significant change in conditions. On the other hand, H2-PHBH did not bind tightly to the Ni-chelate support. H3-PHBH was purified from the crude extract in a single step by using the optimized length and location of the polyhistidine tail in the enzyme.

Physicochemical and immunochemical characterization of salicylate 5-hydroxylase, m-hydroxybenzoate 6-hydroxylase and p-hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis

Salicylate 5-hydroxylase (SAL5H), m-hydroxybenzoate 6-hydroxylase (MHB6H), and p-hydroxybenzoate 3-hydroxylase (PHB3H) from Gram-positive Rhodococcus erythropolis strain S1 were characterized physicochemically and immunochemically. The subunit size and amino acid composition of SAL5H, MHB6H, and PHB3H from strain S1 showed properties similar to those of other flavin-containing aromatic compound monooxygenases such as p-hydroxybenzoate hydroxylase and salicylate 1-hydroxylase (SAL1H), belonging to p-hydroxybenzoate hydroxylase-class, except for homotetrameric structure and cofactor specficity. The N-terminal amino acid sequence of MHB6H from strain S1 indicated significant similarity of ADP-binding region in the N-terminal portion of the enzyme with that known for SAL1H from Pseudomonas putida. Immunochemical properties, determined while conducting serological experiments, showed SAL5H and MHB6H from strain S1 to be immunologically different from PHB3H from strain S1, while SAL5H and MHB6H to apparently share partial antigenic determinants.