Payne Karl A P, Marshall Stephen A, Fisher Karl, Rigby Stephen E J, Cliff Matthew J, Spiess Reynard, Cannas Diego M, Larrosa Igor, Hay Sam, Leys David
Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Department of Chemistry, University of Manchester, Chemistry Building, Oxford Road, Manchester M13 9PL, United Kingdom.
ACS Catal. 2021 Mar 5;11(5):2865-2878. doi: 10.1021/acscatal.0c05042. Epub 2021 Feb 17.
The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the virulence attenuation factor PA0254HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMNimidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on quorum sensing.
可逆（脱）羧酶的UbiD家族的活性依赖于最近发现的异戊二烯化黄素单核苷酸（prFMN）辅因子。模型酶阿魏酸脱羧酶（Fdc1）通过可逆的1,3-环加成过程使不饱和脂肪酸脱羧。蛋白质工程已将Fdc1的底物范围扩展到包括（杂）芳族酸，尽管催化速率仍然很低。这就提出了一个问题，即其他UbiD家族成员如何实现（杂）芳族酸的高效脱羧。在这里，我们表明毒力衰减因子PA0254HudA是一种吡咯-2-羧酸脱羧酶。在存在可逆抑制剂咪唑的情况下，该酶的晶体结构显示形成了共价prFMN-咪唑加合物。底物筛选表明，HudA和选定的活性位点变体可以接受适度范围的杂芳族化合物，包括噻吩-2-羧酸。结合计算研究，我们的数据表明prFMN共价催化通过亲电芳香取代发生，并将HudA活性与吡咯-2-羧酸对群体感应的抑制作用联系起来。
