Seo Pil-Won, Hwangbo Seung-A, Kim Jeong-Sun, Park Suk-Youl
Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
Proteins. 2025 Jul 12. doi: 10.1002/prot.70020.
Homogentisate 1,2-dioxygenase (HGD) is a non-heme iron enzyme that plays a crucial role in phenylalanine and tyrosine metabolism. Acinetobacter-derived HGD (AcHGD) exhibits structural similarity to glyoxalase I (GLO1) but lacks GLO1 activity. In this study, we analyzed the crystal structure of AcHGD at a resolution of 1.5 Å and investigated the molecular basis for its lack of GLO1 activity using enzymatic assays, isothermal titration calorimetry (ITC), and site-directed mutagenesis. Metal ion dependency assays revealed that AcHGD exhibits high specificity for Fe, supporting its role as a non-heme iron (II)-dependent dioxygenase. Structural analysis revealed that AcHGD adopts a β-barrel fold similar to GLO1 and coordinates Zn through a 2-His-1-carboxylate facial triad. However, its substrate-binding tunnel is narrower than that of GLO1, preventing the binding of S-D-lactoylglutathione, the natural substrate of GLO1. Moreover, introducing GLO1-like mutations in the active site failed to confer GLO1 activity and instead abolished HGD activity. ITC analysis confirmed that AcHGD binds strongly to homogentisate but does not interact with S-D-lactoylglutathione. These findings demonstrate that despite its structural resemblance to GLO1, AcHGD lacks GLO1 activity due to differences in substrate specificity and active site architecture. This study provides insights into the structure-function relationship and evolutionary divergence between HGD and GLO1 enzymes.
尿黑酸1,2-双加氧酶（HGD）是一种非血红素铁酶，在苯丙氨酸和酪氨酸代谢中起关键作用。不动杆菌来源的HGD（AcHGD）与乙二醛酶I（GLO1）在结构上相似，但缺乏GLO1活性。在本研究中，我们解析了分辨率为1.5Å的AcHGD晶体结构，并使用酶活性测定、等温滴定量热法（ITC）和定点诱变研究了其缺乏GLO1活性的分子基础。金属离子依赖性测定表明，AcHGD对铁具有高度特异性，支持其作为非血红素铁（II）依赖性双加氧酶的作用。结构分析表明，AcHGD采用与GLO1相似的β桶折叠结构，并通过一个二组氨酸-一羧酸面部三联体配位锌。然而，其底物结合通道比GLO1的窄，阻止了GLO1的天然底物S-D-乳酰谷胱甘肽的结合。此外，在活性位点引入类似GLO1的突变未能赋予其GLO1活性，反而消除了HGD活性。ITC分析证实，AcHGD与尿黑酸强烈结合，但不与S-D-乳酰谷胱甘肽相互作用。这些发现表明，尽管AcHGD与GLO1在结构上相似，但由于底物特异性和活性位点结构的差异，AcHGD缺乏GLO1活性。本研究为HGD和GLO1酶之间的结构-功能关系及进化差异提供了见解。
