An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus flavus

Abstract

SUMMARY The nucleotide sequence of a 1:1 kb Xhol-HindIII fragment downstream of the malate dehydrogenase (mdh) gene of Thermus flavus revealed the presence of an ORF and an incomplete ORF lacking its NH2-terminal portion, in the opposite orientation to that of the mdh gene. These two genes overlapped with each other, sharing two base pairs, suggesting that these genes are co-transcribed in a single mRNA. One ORF (termed gpt) encoded a protein of 154 amino acids showing significant amino acid sequence similarity to purine phosphoribosyltransferases, such as xanthine-guanine phosphoribosyltransferase of Escherichia coli and human hypoxanthine phosphoribosyltransferase. Cloning and sequencing of the upstream region of the gpt gene, together with sequence comparison of the gene product encoded by the region upstream of gpt, suggested that the upstream ORF encoded two in-frame overlapping aspartokinase genes, askA, encoding the β-subunit of 405 amino acids, and askB, encoding the β-subunit of 161 amino acids, which was part of the 3′ portion of askA. Consistent with the sequence data, the askAB and the gpt genes conferred the heat-stable enzyme activities of aspartokinase and phosphoribosyltransferase, respectively, on E. coli. Preliminary characterization of these enzymes produced in E. coli is described.