Nucleotide Sequence and Characterization of the Gene Coding for a Thermostable Heme O Synthase from the Thermophilic Bacillus PS3

The whole caaF-caaE-caaABCD gene cluster, contains two open reading frames (caaE and caaF seem to be coding for the proteins related to heme biosynthesis pathway) on the 5’-upstream of the caa3-type cytochromec oxidase structural genes (caaA, caaB, caaC, and caaD), was isolated from a genomic DNA library of the thermophilic bacterium Bacillus PS3. The caaE gene and its flanking regions were identified. The deduced amino acid sequence of the caaE product is composed of 309 amino acid residues, and is homologous to thatof the cyoE product (heme O synthase) of bo-type ubiquinol oxidase operon from Escherichia coli. The caaE is located on the 5’-upstream of the caaA (corresponds to subunit II of PS3 cytochrome c oxidase) apart from 77 base pairs, and there are a large stem-loop structure and promoter-like structures in this region. This promoter-like structure was needed for the in vivo expression of caaA from PS3 in E. coli. The caaF is located on the 5’-upstream of the caaE apart from 175 base pairs in the reverse direction. A recombinant E. coli cell harboring the caaF-caaE-caaABCD gene cluster of PS3 synthesized heme A. The product of caaE (CaaE) was expressed in E. coli, and had a thermostable heme O synthase (protoheme IX farnesyltransferase) activity in cytoplasmic membrane [Saiki, K., Mogi, T., Ishizuka, M., and Anraku, Y., FEBS Lett. 351(2), 385-388, 1994]. Furthermore, PS3 CaaE amino acid residues as candidates for taking part in prenyl binding, transfer, or structure preserving, and for participating heme B binding or structure preserving by the comparison among CaaE homologues are discussed.【査読有