Plant Immunity Laboratory, National Institute of Plant Genome Research, New Delhi, India.
Rm-403/440, Structural Biology Laboratory, School of Life Science, Jawaharlal Nehru University, New Delhi, India.
FEBS J. 2022 Sep;289(18):5531-5550. doi: 10.1111/febs.16445. Epub 2022 Mar 29.
Old yellow enzymes (OYEs) play a critical role in antioxidation, detoxification and ergot alkaloid biosynthesis processes in various organisms. The yeast- and bacteria-like OYEs have been structurally characterized earlier, however, filamentous fungal pathogens possess a novel OYE class, that is, class III, whose biochemical and structural intricacies remain unexplored to date. Here, we present the 1.6 Å X-ray structure of a class III member, OYE 6 from necrotrophic fungus Ascochyta rabiei (ArOYE6), in flavin mononucleotide (FMN)-bound form (PDB ID-7FEV) and provide mechanistic insights into their catalytic capability. We demonstrate that ArOYE6 exists as a monomer in solution, forms (β/α) barrel structure harbouring non-covalently bound FMN at cofactor binding site, and utilizes reduced nicotinamide adenine dinucleotide phosphate as its preferred reductant. The large hydrophobic cavity situated above FMN, specifically accommodates 12-oxo-phytodienoic acid and N-ethylmaleimide substrates as observed in ArOYE6-FMN-substrate ternary complex models. Site-directed mutations in the conserved catalytic (His196, His199 and Tyr201) and FMN-binding (Lys249 and Arg348) residues render the enzyme inactive. Intriguingly, the ArOYE6 structure contains a novel C-terminus (369-445 residues) made of three α-helices, which stabilizes the FMN binding pocket as its mutation/truncation lead to complete loss of FMN binding. Moreover, the loss of the extended C-terminus does not alter the monomeric nature of ArOYE6. In this study, for the first time, we provide the structural and biochemical insights for a fungi-specific class III OYE homologue and dissect the oxidoreductase mechanism. Our findings hold broad biological significance during host-fungus interactions owing to the conservation of this class among pathogenic fungi, and would have potential implications in the pharmacochemical industry.
老化的黄色酶（OYE）在各种生物体的抗氧化、解毒和麦角生物碱生物合成过程中起着关键作用。酵母和细菌样的 OYE 结构已被早期表征，然而，丝状真菌病原体拥有一种新型的 OYE 类，即 III 类，其生化和结构复杂性迄今尚未得到探索。在这里，我们展示了坏死真菌根腐病菌（Ascochyta rabiei）中 III 类成员 OYE6 的 1.6Å X 射线结构，以黄素单核苷酸（FMN）结合形式（PDB ID-7FEV）呈现，并提供了其催化能力的机制见解。我们证明，ArOYE6 在溶液中以单体形式存在，形成（β/α）桶结构，在辅因子结合位点处含有非共价结合的 FMN，并利用还原型烟酰胺腺嘌呤二核苷酸磷酸作为其首选还原剂。位于 FMN 上方的大疏水性腔，特别容纳了 12-氧代-植物二烯酸和 N-乙基马来酰亚胺底物，如在 ArOYE6-FMN-底物三元复合物模型中观察到的那样。在保守的催化（His196、His199 和 Tyr201）和 FMN 结合（Lys249 和 Arg348）残基处的定点突变使酶失活。有趣的是，ArOYE6 结构包含一个由三个α-螺旋组成的新型 C 端（369-445 残基），它稳定 FMN 结合口袋，因为其突变/截断导致完全丧失 FMN 结合。此外，延伸的 C 端缺失不会改变 ArOYE6 的单体性质。在这项研究中，我们首次为真菌特异性 III 类 OYE 同源物提供了结构和生化见解，并剖析了氧化还原酶机制。由于该类在致病真菌中普遍存在，因此我们的发现在宿主-真菌相互作用中具有广泛的生物学意义，并且在药理学和化工业中可能具有潜在意义。
