Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

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Citation

Quadri LE, Weinreb PH, Lei M, Nakano MM, Zuber P, Walsh CT

Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

Biochemistry. 1998 Feb 10;37(6):1585-95.

PubMed ID
9484229 [ View in PubMed
]
Abstract

The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier 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 carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM 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 carrier 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 microM. 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 carrier 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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Polypeptides
NameUniProt ID
4'-phosphopantetheinyl transferase sfpP39135Details