The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carder protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carder protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, k(cat) values of 56-104 min-1 and K(m) values of 1.3-1.8 μM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carder protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo- synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 μM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carder protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.
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