Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases
Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases
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DOI：
10.1021/bi9719861
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发表时间：
1998-02-10
期刊：
影响因子：
2.9
通讯作者：
Walsh, CT
中科院分区：
文献类型：
--
作者：
Quadri, LEN;Weinreb, PH;Walsh, CT
枯草芽孢杆菌SFP酶是生产脂七肽抗生素表面素所必需的，翻译后使表面素合成酶前三个亚基(SrfABC)的七个肽载体蛋白结构域中的每个丝氨酸残基发生磷酸化，从而产生氨基酸负载和肽键形成的对接位置。以重组SFP和从SrfB1和SrfB2模块中切下的16-17 kDa多肽载体蛋白(PCP)结构域为载脂蛋白底物，测定了56-104min(-1)的k(CAT)值和1.3-1.8muM的K-m值，表明SFP对相邻的PCP结构域具有同等的识别能力。与以前研究的其他磷酸天冬氨酰转移酶(PPTase)不同，SFP将修饰异源重组蛋白的载脂蛋白形式，包括酿酒酵母Lys2的PCP结构域(参与赖氨酸的生物合成)、大肠杆菌EntB的芳基载体蛋白(ARCP)结构域(参与肠道肌动蛋白的生物合成)以及大肠杆菌的酰基载体蛋白(ACP)亚单位，这表明SFP是与多肽和聚酮合成酶基因共表达以过度生产全合成酶的良好候选者。硫代辅酶A和同型半胱胺辅酶A也是SFP的底物，SFP还利用苯甲酰辅酶A和苯乙酰辅酶A，直接将酰基磷酸铁蛋白基团转移到载体蛋白底物中。对PPTase家族中保守的五个残基在SFP中的突变进行了评估，以了解体内对表面素产量的影响和体外对PPTase活性的影响。
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, k(cat) values of 56-104 min(-1) and K-m values of 1.3-1.8 mu M 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 mu M. 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.