Analysis of surfactin synthetase subunits in srfA mutants of Bacillus subtilis OKB105.
The srfA operon of Bacillus subtilis functions in the biosynthesis of the lipopeptide antibiotic surfactin. On the basis of nucleotide sequence and genetic analysis, it is believed to encode three enzymes (E1A, E1B, and E2) that catalyze the incorporation of the surfactin substrate amino acids. Insertion, deletion, and amino acid substitution mutations of srfA were analyzed for subunit composition and activity as determined by assays of both amino acid-dependent ATP-PPi exchange and aminoacyl thioester formation. Insertion mutations in srfAA (encoding E1A, the subunit that incorporates Glu, Leu, and D-Leu) eliminated production and activity of all three enzymes. Deletions within srfAA and extending from srfAA to srfAB (encoding E1B, which incorporates Val, Asp, and D-Leu) abolished the activity and production of all three enzymes. Insertions between srfAA and srfAB and within srfAB eliminate the production and activity of E1B and E2. An insertion mutation in srfAC (encoding E2, which incorporates Leu) abolished the activity of E2 only. Mutations of the active serine in the putative 4'-phosphopantetheine-binding motif of the second and third domains of E1A eliminated thioester formation and severely reduced the ATP-PPi exchange activity of the two domains. However, the same mutation in the first domain of E1B had little effect on Val-dependent ATP-PPi exchange activity but abolished thioester formation. These results indicate that the coding assignments of the srfA genes are srfAA (E1A), srfAB (E1B), and srfAC (E2).
Vollenbroich
D
D
Fachbereich 3 für Chemie und Biotechnologie der Technischen Fachhochschule Berlin, Germany.
Mehta
N
N
Zuber
P
P
Vater
J
J
Kamp
R M
RM
D000215:Acylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D016254:Mutagenesis, Insertional; D009876:Operon / Q000235:genetics*; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D010453:Peptide Synthases; C069542:surfactin synthetase