Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus brevis

• Published in: Journal of biochemistry (Tokyo) 1995-02, Vol.117 (2), p.276-282
• Main Authors: Saito, Masaki, Hori, Kazuko, Kurotsu, Toshitsugu, Kanda, Masayuki, Saito, Yoshitaka
• Format: Article
• Language: English
• Subjects: Amino Acid Isomerases - biosynthesis; Amino Acid Isomerases - chemistry; Amino Acid Isomerases - metabolism; Amino Acid Sequence; aminoacyl adenylation; ATP-binding; Bacillus - enzymology; Bacillus - genetics; Bacillus brevis; Base Sequence; Blotting, Western; Conserved Sequence; DNA Primers; Glycine; gramicidin S synthetase 2; Molecular Sequence Data; Multienzyme Complexes - biosynthesis; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Peptide Synthases - biosynthesis; Peptide Synthases - chemistry; Peptide Synthases - metabolism; peptide synthetase; Point Mutation; Polymerase Chain Reaction; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Species Specificity; Valine
• ISSN: 0021-924X
• DOI: 10.1093/jb/117.2.276

The translated product from the gene fragment containing the second and third domains of gramicidin S synthetase 2 was purified to an essentially homogeneous state. It showed valine- and ornithine-activating activity and the second domain was proved to be the valine-activating domain. Three mutant genes from Bacillus brevis Nagano, BI-3, E-4, and E-5 strains, which encode defective valine-activating domains of gramicidin S synthetase 2, were sequenced. By comparison with the wild-type gene, single point mutations of guanine to adenine were found at the three conserved glycine codons; the 5303rd guanine in BI-3, the 5378th guanine in E-4, and the 4967th guanine in E-5, which corresponded to codon changes of the 1768th glycine to glutamic acid and the 1793rd and the 1656th glycine to aspartic acid. Loss of valine-adenylation activity by mutation at the 1656th glycine proved the direct participation of the TSGT/STGXPKG motif in the adenylation reaction, and suggests that this glycine residue with the conserved lysine residue of the motif forms the phosphate-binding loop for ATP-binding. The 1793rd glycine is a member of the YGXTE motif which was also conserved among adenylate-forming enzymes except acetyl-CoA synthetases. The 1768th glycine residue appears to maintain the conformation of the active site for aminoacyl adenylation since this residue is retained among the adenylate-forming enzymes, though flanking regions are not conserved. These results suggest that these glycine residues are essential for adenylate formation in the antibiotic peptide synthetase family and some other adenylate-forming enzymes.