An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp

Abstract

The nucleotide sequence of the promoter region of the gene for cholesterol oxidase (choA) from Streptomyces sp. strain SA-COO was determined. We found an open reading frame (choP) that is located between a potential promoter sequence and the structural gene for the ChoA protein. Deletion analysis showed that the promoter region for choP is essential for expression of the choA gene. Mappings of S1 nuclease and primer extension of transcripts generated in vivo suggested that the synthesis of mRNA starts at a site 41 bases upstream from the ATG initiation codon of the choP gene. By Northern (RNA) blot analysis of the transcripts, we found a 2.9-kilobase transcript that is identical in size to the total sequence of the choP and choA genes. These results suggest that the two genes, choP and choA, are transcribed polycistronically under the control of the promoter that is upstream from the structural gene for choP. The choP gene encodes a protein of 381 amino acids with a calculated Mr of 41,668. The nucleotide sequence of the choP gene has a high degree of similarity to the sequence of the genes for cytochrome P-450s from humans and Pseudomonas species. A region of homology with the cytochrome P-450s from various organisms was identified in the choP protein and may represent a region associated with a binding site for heme iron. Analysis of the CO difference spectrum of an extract of Streptomyces lividans cells that carry a plasmid which includes the choP gene revealed a unique peak, characteristic of cytochrome P-450, which is identical to that obtained with the parent strain.