Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein.
In this paper, the initiation reactions in surfactin biosynthesis by Bacillus subtilis OKB 105 were investigated. Evidence for a specific role of the SrfD protein, the external thioesterase enzyme in surfactin biosynthesis, was obtained for the first time. The action of SrfD was investigated both with the native, but only partially purified, enzyme and the highly purified, His-tagged protein overexpressed in Escherichia coli. Surfactin can be formed by the interaction of the three amino acid activating components of surfactin synthetase SrfA, B and C alone. This process is stimulated by SrfD. In the initiation reactions, the beta-hydroxy fatty acid substrate is transferred from beta-hydroxymyristoyl-coenzyme A to the start enzyme SrfA followed by formation of beta-hydroxymyristoyl-glutamate. The same reactions were also observed with the recombinant L-Glu-activating module of surfactin synthetase. Lipopeptide formation can be initiated by these function units alone, but SrfD efficiently supports and stimulates the formation of initiation products. From these results, we infer that SrfD functions as the thioesterase/acyltransferase enzyme in the initiation process previously postulated by Menkhaus et al. [Menkhaus et al. (1993) J. Biol. Chem. 268, 7678-7684], thus enhancing surfactin formation.
Steller
Sigrid
S
Institut für Chemie, Arbeitsgruppe Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin, Germany.
Sokoll
Andrea
A
Wilde
Christopher
C
Bernhard
Frank
F
Franke
Peter
P
Vater
Joachim
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D049148:Aminoacylation; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology; D003065:Coenzyme A / Q000737:chemistry; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D055666:Lipopeptides; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology; D008969:Molecular Sequence Data; D009227:Myristic Acids / Q000737:chemistry; D010442:Peptide Chain Initiation, Translational; D010453:Peptide Synthases / Q000737:chemistry / Q000502:physiology; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000502:physiology*
D001426:Bacterial Proteins; D055666:Lipopeptides; D008074:Lipoproteins; D009227:Myristic Acids; D010456:Peptides, Cyclic; D021122:Protein Subunits; C009893:beta-hydroxymyristic acid; C009365:surfactin peptide; D018698:Glutamic Acid; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; D003065:Coenzyme A