Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli.
Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.
Ju
Jiansong
J
Department of Applied Bioresource Science, The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.
Yokoigawa
Kumio
K
Misono
Haruo
H
Ohnishi
Kouhei
K
D000411:Alanine Racemase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D023061:Gene Order; D005798:Genes, Bacterial / Q000235:genetics*; D009154:Mutation; D010802:Phylogeny; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011551:Pseudomonas fluorescens / Q000145:classification / Q000201:enzymology* / Q000235:genetics
D021122:Protein Subunits; D000411:Alanine Racemase