Studies on gramicidin S synthetase. Purification of the heavy enzyme obtained from some mutants of Bacillus brevis.
The heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20,W value of 12.2 at a protein concentration of 2.5 mg per ml. These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.
D000591:Amino Acid Isomerases / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002499:Centrifugation, Density Gradient / Q000379:methods; D006096:Gramicidin / Q000096:biosynthesis; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification*; D009154:Mutation; D010453:Peptide Synthases / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship
D009097:Multienzyme Complexes; D006096:Gramicidin; D000591:Amino Acid Isomerases; D010453:Peptide Synthases