3 CONSERVED GLYCINE RESIDUES IN VALINE ACTIVATION OF GRAMICIDIN-S SYNTHETASE-2 FROM BACILLUS-BREVIS

The translated product from the gene fragment containing the second an d third domains of gramicidin S synthetase 2 was purified to an essent ially homogeneous state, It showed valine- and ornithine-activating ac tivity and the second domain was proved to be the valine-activating do main. Three mutant genes from Bacillus brevis Nagano, BI-3, E-4, and E -5 strains, which encode defective valine-activating domains of gramic idin S synthetase 2, were sequenced. By comparison with the wild-type gene, single point mutations of guanine to adenine were found at the t hree 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 c odon changes of the 1768th glycine to glutamic acid and the 1793rd and the 1656th glycine to aspartic acid. Loss of valine-adenylation activ ity by mutation at the 1656th glycine proved the direct participation of the TSGT/STGXPKG motif in the adenylation reaction, and suggests th at 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 adenylat e-forming enzymes except acetyl-CoA synthetases. The 1768th glycine re sidue appears to maintain the conformation of the active site for amin oacyl adenylation since this residue is retained among the adenylate-f orming enzymes, though flanking regions are not conserved, These resul ts suggest that these glycine residues are essential for adenylate for mation in the antibiotic peptide synthetase family and some other aden ylate-forming enzymes,