Max-Planck Unit for Structural Molecular Biology, MPG-ASMB c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.
J Mol Biol. 2010 Feb 26;396(3):785-99. doi: 10.1016/j.jmb.2009.12.009. Epub 2009 Dec 16.
The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.
阿氏节杆菌中尼古丁氧化降解途径包括两种在基因和结构上均无关联的黄素酶，即 6-羟基-L-烟碱氧化酶（6HLNO）和 6-羟基-D-烟碱氧化酶，它们分别对 L-和 D-型 6-羟基烟碱具有绝对立体特异性。我们通过同晶置换/多波长反常散射相位解析法解析了 6HLNO 的晶体结构，分辨率为 1.95Å。6HLNO 同源二聚体每个亚基的整体结构和各个结构域的折叠与真核单胺氧化酶非常相似。出乎意料的是，发现一个二酰基甘油磷脂分子非共价结合到每个 6HLNO 单体上。脂肪酸链占据贯穿每个亚基底物结合域内部的疏水性通道。暴露于溶剂的甘油磷酸部分位于亚基-亚基界面处。我们进一步解析了二硫代苏糖醇还原的 6HLNO 与天然底物 6-羟基-L-烟碱复合物的晶体结构，分辨率为 2.05Å。在一个紧密的腔室中定位的底物表明，这个非生产性复合物的结合几何形状可能与氧化条件下非常相似。相对于黄素腺嘌呤二核苷酸辅因子的异咯嗪环，观察到的结合底物的取向适合于在形成底物分子手性中心的碳原子上进行氢转移脱氢。基于与 D-底物复合物的模型，比较 6HLNO 和 6-羟基-D-烟碱氧化酶的底物结合模式，为两种酶的立体特异性提供了一种解释。提议两种酶将对映体底物相对于黄素平面以镜像对称的方式取向。
