THE NUCLEOTIDE-SEQUENCE FOR A PROLINE-ACTIVATING DOMAIN OF GRAMICIDIN-S SYNTHETASE-2 GENE FROM BACILLUS-BREVIS

A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-(PP(i))-P-32 exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end. A comparison of the putative amino acid sequence of grs 2-pro gene product showed that the amino-terminal region was highly homologous to the available sequence of tyrocidine synthetase 2 (71% similarity) and the carboxy-terminal region of the gene product had significant homology to the amino-terminal half of GS 1 (66% similarity), as well as to other antibiotic peptide synthetases, tyrocidine synthetase 1 and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), an enzyme involved in penicillin biosynthesis.