Richardson Shona M, Harrison Peter J, Herrera Michael A, Wang Menglu, Verez Rebecca, Ortiz Gustavo Perez, Campopiano Dominic J
School of Chemistry University of Edinburgh, David Brewster Road, Edinburgh, EH9 3FJ, UK.
Diamond Light Source Ltd., Harwell Science & innovation Campus, Didcot, OX11 0DE, UK.
Chembiochem. 2022 Sep 5;23(17):e202200171. doi: 10.1002/cbic.202200171. Epub 2022 Jul 13.
The carbon backbone of biotin is constructed from the C di-acid pimelate, which is converted to an acyl-CoA thioester by an ATP-dependent, pimeloyl-CoA synthetase (PCAS, encoded by BioW). The acyl-thioester is condensed with ʟ-alanine in a decarboxylative, Claisen-like reaction to form an aminoketone (8-amino-7-oxononanoic acid, AON). This step is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent enzyme (AON synthase, AONS, encoded by BioF). Distinct versions of Bacillus subtilis BioW (BsBioW) and E. coli BioF (EcBioF) display strict substrate specificity. In contrast, a BioW-BioF fusion from Corynebacterium amycolatum (CaBioWF) accepts a wider range of mono- and di-fatty acids. Analysis of the active site of the BsBioW : pimeloyl-adenylate complex suggested a key role for a Phe (F192) residue in the CaBioW domain; a F192Y mutant restored the substrate specificity to pimelate. This surprising substrate flexibility also extends to the CaBioF domain, which accepts ʟ-alanine, ʟ-serine and glycine. Structural models of the CaBioWF fusion provide insight into how both domains interact with each other and suggest the presence of an intra-domain tunnel. The CaBioWF fusion catalyses conversion of various fatty acids and amino acids to a range of AON derivatives. Such unexpected, natural broad substrate scope suggests that the CaBioWF fusion is a versatile biocatalyst that can be used to prepare a number of aminoketone analogues.
生物素的碳骨架由C二酸庚二酸构建而成，庚二酸通过一种依赖ATP的庚二酸单酰辅酶A合成酶（PCAS，由BioW编码）转化为酰基辅酶A硫酯。酰基硫酯在一个脱羧的、类似克莱森缩合的反应中与L-丙氨酸缩合，形成一种氨基酮（8-氨基-7-氧代壬酸，AON）。这一步由依赖磷酸吡哆醛（PLP）的酶（AON合酶，AONS，由BioF编码）催化。枯草芽孢杆菌BioW（BsBioW）和大肠杆菌BioF（EcBioF）的不同版本表现出严格的底物特异性。相比之下，来自无枝菌酸棒杆菌的BioW-BioF融合蛋白（CaBioWF）能接受更广泛的单脂肪酸和二脂肪酸。对BsBioW：庚二酸单酰腺苷酸复合物活性位点的分析表明，CaBioW结构域中的苯丙氨酸（F192）残基起关键作用；F192Y突变体将底物特异性恢复为庚二酸。这种惊人的底物灵活性也延伸到了CaBioF结构域，该结构域能接受L-丙氨酸、L-丝氨酸和甘氨酸。CaBioWF融合蛋白的结构模型有助于深入了解两个结构域如何相互作用，并表明存在一个结构域内通道。CaBioWF融合蛋白催化各种脂肪酸和氨基酸转化为一系列AON衍生物。这种意想不到的天然广泛底物范围表明，CaBioWF融合蛋白是一种通用的生物催化剂，可用于制备多种氨基酮类似物。
