Quadri L E, Weinreb P H, Lei M, Nakano M M, Zuber P, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1998 Feb 10;37(6):1585-95. doi: 10.1021/bi9719861.
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.
枯草芽孢杆菌的Sfp酶是脂肽抗生素表面活性素产生所必需的，它能在翻译后将磷酸泛酰巯基乙胺基连接到表面活性素合成酶前三个亚基（SrfABC）的七个肽基载体蛋白结构域中每个结构域的一个丝氨酸残基上，从而产生用于氨基酸加载和肽键形成的对接位点。以重组Sfp和从SrfB1和SrfB2模块切除的16 - 17 kDa肽基载体蛋白（PCP）结构域作为脱辅基底物，测定的kcat值为56 - 104 min-1，K(m)值为1.3 - 1.8 microM，表明Sfp对相邻PCP结构域具有同等识别能力。与之前研究过的其他磷酸泛酰巯基乙胺基转移酶（PPTases）不同，Sfp能修饰异源重组蛋白的脱辅基形式，包括酿酒酵母Lys2的PCP结构域（参与赖氨酸生物合成）、大肠杆菌EntB的芳基载体蛋白（ArCP）结构域（参与肠杆菌素生物合成）以及大肠杆菌酰基载体蛋白（ACP）亚基，这表明Sfp是与肽和聚酮合酶基因进行异源共表达以过量生产全酶的良好候选者。共底物辅酶A（CoA）作为磷酸泛酰巯基乙胺基的供体，其K(m)为0.7 microM。脱硫CoA和高半胱胺CoA也是Sfp的底物，苯甲酰CoA和苯乙酰CoA也能被Sfp利用，从而导致酰基磷酸泛酰巯基乙胺基部分直接转移到载体蛋白底物中。对PPTase家族中保守的五个残基在Sfp中进行诱变，评估其对表面活性素产生的体内影响以及对PPTase活性的体外影响。
