Entire nucleotide sequence for Bacillus brevis Nagano Grs2 gene encoding gramicidin S synthetase 2: a multifunctional...

Bacillus brevis Nagano grs2 gene, which encodes gramicidin S synthetase 2 (GS2) catalyzing activation and combination of four constituent amino acids of gramicidin S, namely, proline, valine, ornithine, and leucine, has been sequenced. The open reading frame of grs2 gene specifies a 4,450-amino acid protein with a calculated molecular weight of 508,658. There are four domains with a mean of 1,042 amino acid residues containing a repeated sequence of about 600 amino acids, which is highly homologous to the amino-terminal half of gramicidin S synthetase 1 (GS1) (about 40-50% identity). Three domains of grs2 protein, excluding the first one, show homology over the entire sequences of 1,042 amino acids, but the first domain only shows homology in the conserved 600-amino acid sequence. The last 300-amino acid sequence of grs2 protein following the fourt domain has no homology with any of the above sequences. Translation products of subcloned fragments containing the third or the fourth domain catalyzed ornithine- or leucine-dependent ATP-32Pi exchange, respectively. These results, together with a previous report on a proline-activation domain indicated that the repeated and conserved domains are the individual activation sites of the constituent amino acids; the activation sites are arranged in the order of peptide elongation on GS2. Several motifs of grs2 protein are conserved among the multiple domains of peptide synthetases and aminoacyl or acyl adenylate-forming enzymes.