Effect of single base substitutions at glycine-870 codon of gramicidin S synthetase 2 gene on proline activation.
The mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation. By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid. Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: alpha-aminoadipate reductase (LYS2), EntF, and AngR. On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase. Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis. Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan. The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction. The coil structure of 870-Gly region was lost in the mutants. These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthetase family.
Tokita
K
K
Department of Biochemistry, Hyogo College of Medicine.
Hori
K
K
Kurotsu
T
T
Kanda
M
M
Saito
Y
Y
D000591:Amino Acid Isomerases / Q000235:genetics*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D017124:Conserved Sequence; D004926:Escherichia coli; D005998:Glycine / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics*; D010957:Plasmids; D011392:Proline / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D003062:Codon; D009097:Multienzyme Complexes; D011392:Proline; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D005998:Glycine