Tsujino Shuhei, Yamada Yusuke, Senda Miki, Nakamura Akihiko, Senda Toshiya, Fujiwara Taketomo
Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
Department of Environment and Energy Systems, Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan.
J Bacteriol. 2025 Feb 20;207(2):e0034224. doi: 10.1128/jb.00342-24. Epub 2025 Jan 8.
Nitrification by heterotrophic microorganisms is an important part of the nitrogen cycle in the environment. The enzyme responsible for the core function of heterotrophic nitrification is pyruvic oxime dioxygenase (POD). POD is a non-heme, Fe(II)-dependent enzyme that catalyzes the dioxygenation of pyruvic oxime to produce pyruvate and nitrite. To analyze the catalytic mechanism of POD, the crystal structure of POD from (AfPOD) was determined at 1.76 Å resolution. The enzyme is a homotetramer, and the subunit structure is homologous to those of class II aldolases, in particular, a zinc-dependent L-fuculose-1-phosphate aldolase. The active site of the subunit is located at the bottom of a cleft formed with an adjacent subunit. The iron ion at the active site is coordinated by three histidines and three water molecules in an octahedral geometry. The putative oxygen tunnel was connected between the active site and the central cavity of the tetramer. The N-terminal region of AfPOD, which is essential for catalytic activity, is disordered in the crystal. Structure prediction with AlphaFold2 combined with mutational experiments suggested that the disordered N-terminal region adopts an α-helix conformation and participates in the formation of the active site. The catalytic mechanism of the dioxygenase reaction by POD is discussed on the basis of the molecular docking model.IMPORTANCEOur knowledge of nitrification has increased considerably in recent decades with the discovery of new nitrifying microorganisms and the characterization of their biochemical processes. Some heterotrophic bacteria and fungi are known to show nitrification activities, but the molecular mechanisms have been poorly understood. Here, we performed a structural characterization of pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification that produces nitrite from ammonia using pyruvic oxime as an intermediate. Structural and enzymatic analyses revealed that POD is a unique dioxygenase with features such as an aldolase backbone, an N-terminal α-helix, and an oxygen tunnel. Our results provide insights not only into the molecular mechanisms but also into the design of specific inhibitors of heterotrophic nitrification.
异养微生物的硝化作用是环境中氮循环的重要组成部分。负责异养硝化核心功能的酶是丙酮酸肟双加氧酶（POD）。POD是一种非血红素、依赖Fe(II)的酶，催化丙酮酸肟的双加氧反应生成丙酮酸和亚硝酸盐。为了分析POD的催化机制，测定了嗜碱栖热放线菌（AfPOD）的POD晶体结构，分辨率为1.76 Å。该酶是同四聚体，其亚基结构与II类醛缩酶的亚基结构同源，特别是与锌依赖性L-岩藻糖-1-磷酸醛缩酶同源。亚基的活性位点位于与相邻亚基形成的裂隙底部。活性位点的铁离子由三个组氨酸和三个水分子以八面体几何构型配位。推测的氧通道连接活性位点和四聚体的中央腔。AfPOD的N端区域对催化活性至关重要，在晶体中是无序的。结合突变实验的AlphaFold2结构预测表明，无序的N端区域采用α-螺旋构象并参与活性位点的形成。基于分子对接模型讨论了POD双加氧酶反应的催化机制。
重要性
近几十年来，随着新的硝化微生物的发现及其生化过程的表征，我们对硝化作用的认识有了显著提高。已知一些异养细菌和真菌具有硝化活性，但分子机制尚不清楚。在此，我们对丙酮酸肟双加氧酶（POD）进行了结构表征，POD是异养硝化作用中的关键酶，它以丙酮酸肟为中间体将氨转化为亚硝酸盐。结构和酶学分析表明，POD是一种独特的双加氧酶，具有醛缩酶骨架、N端α-螺旋和氧通道等特征。我们的结果不仅为分子机制提供了见解，也为异养硝化作用的特异性抑制剂的设计提供了见解。
