Heyes Derren J, Lakavath Balaji, Hardman Samantha J O, Sakuma Michiyo, Hedison Tobias M, Scrutton Nigel S
Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
ACS Catal. 2020 Jun 19;10(12):6691-6696. doi: 10.1021/acscatal.0c01684. Epub 2020 May 19.
Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to characterize a red-shifted flavin intermediate observed in the catalytic cycle of FAP. We show that this intermediate can form below the "glass transition" temperature of proteins, whereas the subsequent decay of the species proceeds only at higher temperatures, implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.
脂肪酸光脱羧酶（FAP）是生物燃料和精细化学品生产中一个很有前景的靶点。它含有一个黄素腺嘌呤二核苷酸辅因子，催化脂肪酸的蓝光依赖性脱羧反应，生成相应的烷烃。然而，关于FAP的催化机制，或者光如何用于驱动酶促脱羧反应，人们了解得很少。在这里，我们结合了时间分辨和低温捕获紫外可见吸收光谱，来表征在FAP催化循环中观察到的一个红移黄素中间体。我们表明，这个中间体可以在蛋白质的“玻璃化转变”温度以下形成，而该物种随后的衰减仅在较高温度下进行，这意味着蛋白质运动在中间体的衰减中起作用。溶剂同位素效应测量，结合对FAP选定的定点变体的分析，表明红移黄素物种的形成与来自附近活性位点半胱氨酸残基的氢原子转移直接相关，产生最终的烷烃产物。我们的研究表明，这个半胱氨酸残基形成了一个硫醇盐-黄素电荷转移物种，它被指定为红移黄素中间体。总之，我们的数据为FAP催化的光依赖性脱羧酶机制提供了见解，并突出了基于黄素的光酶（重新）设计中的重要考虑因素。
