Center for Structural Biology and Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.
PLoS One. 2011;6(10):e26021. doi: 10.1371/journal.pone.0026021. Epub 2011 Oct 6.
4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined.
METHODOLOGY/PRINCIPAL FINDINGS: In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding.
CONCLUSIONS/SIGNIFICANCE: The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored.
4-羟基-2-氧代戊二酸（HOG）醛缩酶是羟脯氨酸降解途径中一种独特的酶，可催化 HOG 裂解为丙酮酸和乙醛酸。尽管没有实验数据支持这一假说，但人们认为该酶的突变与原发性高草酸尿症 3 型（PH3）中草酸盐的过度产生有关。此外，人 HOGA 的身份、寡聚状态、酶活性和晶体结构尚未通过实验确定。
方法/主要发现：在这项研究中，通过从牛肾中纯化的线粒体酶的质谱鉴定了人 HOGA（hHOGA）。hHOGA 进行类似于三聚体 2-酮-3-脱氧-6-磷酸葡萄糖醛酸醛缩酶的反醛缩裂解反应。然而，序列比较表明，HOGA 与四聚体细菌二氢二吡啶羧酸合酶有关，但反应方向相反。测定了与丙酮酸结合的 hHOGA 的 1.97Å分辨率晶体结构，使 HOG-Schiff 碱中间产物的建模和活性位点残基的鉴定成为可能。定点突变的动力学分析支持 Lys196 作为亲核试剂、Tyr168 和 Ser77 作为质子传递的组成部分以及 Asn78 和 Ser198 作为促进底物结合的独特残基的重要性。
结论/意义：所提出的生化和结构数据支持 hHOGA 利用 I 型醛缩酶反应机制，但采用了新型残基相互作用来结合底物。PH3 突变的映射确定了活性位点或四聚体组装中潜在的重排，这可能导致活性丧失。总之，这些数据为评估 hHOGA 的突变形式以及如何通过药理学恢复其活性奠定了基础。
