Three conserved glycine residues in valine activation of gramicidin S synthetase 2 from Bacillus brevis.
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.
Saito
M
M
Department of Biochemistry, Hyogo College of Medicine.
Hori
K
K
Kurotsu
T
T
Kanda
M
M
Saito
Y
Y
D000591:Amino Acid Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D017931:DNA Primers; D005998:Glycine; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014633:Valine
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D014633:Valine; D005998:Glycine