CLONING, MAPPING AND FUNCTIONAL-CHARACTERIZATION OF THE HEMB GENE OF PSEUDOMONAS-AERUGINOSA, WHICH ENCODES A MAGNESIUM-DEPENDENT 5-AMINOLEVULINIC ACID DEHYDRATASE

During tetrapyrrole biosynthesis 5-amino-levulinic acid dehydratase (A LAD) catalyzes the condensation of two molecules of 5-aminolevulinic a cid (ALA) to form one molecule of the pyrrole derivative porphobilinog en. In Escherichia coli, the enzyme is encoded by the gene hemB. The h emB gene was cloned from Pseudomonas aeruginosa by functional compleme ntation of an E. coli hemB mutant. An open reading frame of 1011 bp en coding a protein of 336 amino acids (M-r 37008) was identified. The ge ne was mapped to SpeI fragment G and DpnI fragment G of the P. aerugin osa chromosome, corresponding to the 10 to 12 min region of the new ma p or 19 to 22 min interval of the old map, The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma(70)-d ependent promoter were localized. No obvious regulation of the hemB ge ne by oxygen, nitrate, heme or iron was detected. Alignment of the ami no acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg2+-dependent. P. aeruginosa h emB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg2+-dependent activity was directly demons trated.