Lountos George T, Jiang Rongrong, Wellborn William B, Thaler Tracey L, Bommarius Andreas S, Orville Allen M
School of Chemistry and Biochemistry, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Biochemistry. 2006 Aug 15;45(32):9648-59. doi: 10.1021/bi060692p.
The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.
来自旧金山乳杆菌的黄素腺嘌呤二核苷酸（FAD）依赖性烟酰胺腺嘌呤二核苷酸（磷酸）（NAD(P)H）氧化酶（L.san-Nox2）催化2当量的NADH或NADPH的氧化，并将1当量的O₂还原生成2当量的水。在稳态周转期间，溶液中仅检测到0.5%的还原当量以过氧化氢形式存在，这表明在第一个当量的NAD(P)H氧化并与O₂反应后，它不会从酶中释放出来。在此，我们报道了L.san-Nox2的晶体结构，分辨率为1.8 Å。该酶以二聚体形式结晶，每个单体由一个FAD结合结构域（第1 - 120位残基）、一个NAD(P)H结合结构域（第150 - 250位残基）和一个二聚化结构域（第325 - 451位残基）组成。位于FAD si面附近的氧化还原活性半胱氨酸42（Cys42）残基的电子密度与氧化为亚磺酸（Cys-SOH）状态一致。Cys42的侧链也观察到两种构象；一种构象中，亚磺酸与组氨酸10形成氢键，另一种构象中，它与FAD的O2'原子形成氢键。令人惊讶的是，通过电子密度图和对热变性酶释放的配体进行基质辅助激光解吸电离飞行时间（MALDI-TOF）分析确定，每个NAD(P)H结合结构域都含有一个ADP配体。ADP配体与酶共纯化，并且其存在并不抑制酶活性。因此，我们推测NADPH或NADH底物通过一个从酶外部延伸并在FAD re面终止的长通道结合。还利用L.san-Nox2的晶体结构生成了乳酸乳球菌NADH氧化酶（L.lac-Nox2）的同源模型，该模型揭示了这两种酶之间的几个重要异同点。对变性L.lac-Nox2释放的配体进行高效液相色谱（HPLC）分析表明，它不结合ADP，这与该酶对NADH氧化的特异性相关。
