Pant Kartikeya, Bilwes Alexandrine M, Adak Subrata, Stuehr Dennis J, Crane Brian R
The Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 2002 Sep 17;41(37):11071-9. doi: 10.1021/bi0263715.
Eukaryotic nitric oxide synthases (NOSs) produce nitric oxide to mediate intercellular signaling and protect against pathogens. Recently, proteins homologous to mammalian NOS oxygenase domains have been found in prokaryotes and one from Bacillus subtilis (bsNOS) has been demonstrated to produce nitric oxide [Adak, S., Aulak, K. S., and Stuehr, D. J. (2002) J. Biol. Chem. 277, 16167-16171]. We present structures of bsNOS complexed with the active cofactor tetrahydrofolate and the substrate L-arginine (L-Arg) or the intermediate N(omega)-hydroxy-L-arginine (NHA) to 1.9 or 2.2 A resolution, respectively. The bsNOS structure is similar to those of the mammalian NOS oxygenase domains (mNOS(ox)) except for the absence of an N-terminal beta-hairpin hook and zinc-binding region that interact with pterin and stabilize the mNOS(ox) dimer. Changes in patterns of residue conservation between bacterial and mammalian NOSs correlate to different binding modes for pterin side chains. Residue conservation on a surface patch surrounding an exposed heme edge indicates a likely interaction site for reductase proteins in all NOSs. The heme pockets of bsNOS and mNOS(ox) recognize L-Arg and NHA similarly, although a change from Val to Ile beside the substrate guanidinium may explain the 10-20-fold slower dissociation of product NO from the bacterial enzyme. Overall, these structures suggest that bsNOS functions naturally to produce nitrogen oxides from L-Arg and NHA in a pterin-dependent manner, but that the regulation and purpose of NO production by NOS may be quite different in B. subtilis than in mammals.
真核生物一氧化氮合酶（NOSs）产生一氧化氮以介导细胞间信号传导并抵御病原体。最近，在原核生物中发现了与哺乳动物NOS加氧酶结构域同源的蛋白质，并且已证明来自枯草芽孢杆菌的一种蛋白质（bsNOS）能产生一氧化氮[阿达卡，S.，奥拉克，K. S.，和斯图尔，D. J.（2002年）《生物化学杂志》277卷，16167 - 16171页]。我们展示了bsNOS分别与活性辅因子四氢叶酸和底物L - 精氨酸（L - Arg）或中间体N（ω） - 羟基 - L - 精氨酸（NHA）形成的复合物的结构，分辨率分别为1.9埃或2.2埃。bsNOS的结构与哺乳动物NOS加氧酶结构域（mNOS(ox)）的结构相似，只是缺少与蝶呤相互作用并稳定mNOS(ox)二聚体的N端β - 发夹钩和锌结合区域。细菌和哺乳动物NOS之间残基保守模式的变化与蝶呤侧链的不同结合模式相关。围绕暴露血红素边缘的表面斑块上的残基保守性表明这可能是所有NOS中还原酶蛋白的一个相互作用位点。bsNOS和mNOS(ox)的血红素口袋对L - Arg和NHA的识别方式相似，尽管底物胍基旁边从缬氨酸到异亮氨酸的变化可能解释了产物NO从细菌酶上解离的速度慢10 - 20倍的原因。总体而言，这些结构表明bsNOS的天然功能是以蝶呤依赖的方式从L - Arg和NHA产生氮氧化物，但枯草芽孢杆菌中NOS产生NO的调节和目的可能与哺乳动物有很大不同。
