Payne Karl A P, Marshall Stephen A, Fisher Karl, Cliff Matthew J, Cannas Diego M, Yan Cunyu, Heyes Derren J, Parker David A, Larrosa Igor, Leys David
Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
School of Chemistry, University of Manchester, Chemistry Building, Oxford Road, Manchester M13 9PL, U.K.
ACS Catal. 2019 Apr 5;9(4):2854-2865. doi: 10.1021/acscatal.8b04862. Epub 2019 Feb 15.
The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO levels in vitro. We report the crystal structure of a thermophilic HmfF from , revealing that the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrole compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors and improve our understanding of catalysis by members of the UbiD enzyme family.
2,5-呋喃二甲酸（FDCA）的生物合成具有重要价值，它有可能替代石化衍生的单体，如用于聚对苯二甲酸乙二酯（PET）塑料的对苯二甲酸酯。HmfF属于异戊烯化黄素（prFMN）依赖性UbiD家族可逆（脱）羧酶的一个未被表征的分支，据推测它在体内可将2,5-呋喃二甲酸（FDCA）转化为糠酸。我们对HmfF进行了详细表征，并证明HmfF在体外高CO水平下可催化糠酸羧化。我们报道了来自嗜热栖热菌的HmfF的晶体结构，揭示位于prFMN辅因子上方的活性位点包含一个由UbiD酶HmfF分支特有的H296 - R304 - R331残基组成的糠酸/FDCA结合位点。后者的变体活性受损，而H296N改变了对吡咯化合物的底物偏好。对该酶工程化二聚体形式的溶液研究和晶体结构测定揭示了UbiD家族广泛保守的亮氨酸残基在活性中发挥了意想不到的关键作用。对底物和辅因子结合的结构见解为在FDCA塑料前体生产中进一步开发HmfF提供了模板，并增进了我们对UbiD酶家族成员催化作用的理解。
