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.

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.

Bacillus krulwichiae sp. nov., a halotolerant obligate alkaliphile that utilizes benzoate and m-hydroxybenzoate.

Obligate alkaliphilic strains, AM31D(T) and AM11D, that utilize benzoate and m-hydroxybenzoate were isolated from soil obtained from Tsukuba, Ibaraki, Japan. The isolates grew at pH 8-10, but not at neutral pH. They were Gram-positive, facultatively anaerobic, straight rods with peritrichous flagella and produced ellipsoidal spores.