The Rhodobacter capsulatushemA Region: Nucleotide Sequence and Functional Analysis.

Abstract

Most of the effort to elucidate the mechanism of oxygen-mediated regulation of tetrapyrrole biosynthesis in Rhodobacter capsulatus has focused on the first step in the biosynthetic pathway, the condensation of glycine with succinyl-CoA to form $\delta$-aminolevulinic acid. This reaction, in R. capsulatus, is catalyzed by $\delta$-aminolevulinate synthase, the product of the hemA gene. In order to facilitate further study of the hemA gene and its product, its nucleotide sequence was determined. The open reading frame encoding $\delta$-aminolevulinate synthase was determined to be 1,206 base pairs, encoding a 50,491 Da polypeptide of 401 amino acids. The DNA sequence was compared with other hemA genes and found to be between 60% and 70% identical. Likewise, the predicted amino acid sequence of this open reading frame was found to be 70% similar to other $\delta$-aminolevulinate synthases. $\delta$-Aminolevulinate synthase is a pyridoxal phosphate-requiring enzyme; thus, by comparing its amino acid sequence with that of other pyridoxal phosphate-requiring enzymes, a putative pyridoxal phosphate binding site has been identified. When attempts were made to isolate a hemA$\sp-$ strain of R. capsulatus by Tn5 mutagenesis, no complete block in the hemA gene was obtained. Sequence data from the only aminolevulinate-requiring mutant revealed that the Tn5 inserted 20 bases into the coding region for $\delta$-aminolevulinate synthase. Transcription and translation initiating in Tn5, and continuing into the R. capsulatus DNA would account for the low level $\delta$-aminolevulinate synthase activity observed in the mutant. This transcription could be necessary if hemA is part of an operon, and a complete block of transcription from the hemA promoter would have a polar effect on a downstream gene. Sequence data collected reveals that there are two open reading frames downstream of hemA. Both are transcribed in the same direction as hemA. The first begins 84 base pairs past the termination codon for hemA and extends for 1,239 base pairs. The product of this open reading frame has not been identified. Beginning 55 base pairs past the termination codon for orf-2 there is another unidentified open reading frame. The sequences of orf-2 and orf-3 were compared with all sequences in Genbank; no significant homology exists with any other gene.