Gatto Gregory J, McLoughlin Shaun M, Kelleher Neil L, Walsh Christopher T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2005 Apr 26;44(16):5993-6002. doi: 10.1021/bi050230w.
Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.
雷帕霉素、FK506和FK520是由链霉菌中的混合聚酮合酶（PKS）/非核糖体肽合成酶（NRPS）系统产生的强效免疫抑制天然产物大环化合物。这些分子中的一个重要功能元件是L-哌啶酸部分，它通过RapP/FkbP（一种四结构域NRPS）的作用被并入完整的聚酮链中，RapP/FkbP在氨基酸插入后还可能起到环化该链的作用。在此，我们报告了从FK520生物合成途径中表达和纯化重组FkbP的过程。通过结合放射性测定和傅里叶变换质谱，我们证明，一旦FkbP在体外被磷酸泛酰巯基乙胺化，其肽基载体蛋白结构域就能成功装载L-哌啶酸，并在较小程度上装载L-脯氨酸。FkbP的第一个缩合结构域通过使用来自PKS阵列的适当装载的末端酰基载体蛋白FkbA作为链供体，成功地将氨酰基-S-FkbP乙酰化，从而显示出活性。定点诱变证实N端缩合结构域催化转移反应。脯氨酰-S-FkbP的乙酰化比哌啶酰-S-FkbP更快且程度更高，这种趋势在使用替代酰基链供体时也观察到。这些观察结果表明，FkbP的腺苷化结构域是哌啶酸掺入的主要选择性过滤器。
