Crystal Structure Analysis of Free and Substrate-Bound 6-Hydroxy-L-Nicotine Oxidase from Arthrobacter nicotinovorans
Crystal Structure Analysis of Free and Substrate-Bound 6-Hydroxy-L-Nicotine Oxidase from Arthrobacter nicotinovorans
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DOI：
10.1016/j.jmb.2009.12.009
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发表时间：
2010-02-26
影响因子：
5.6
通讯作者：
Bartunik, Hans D.
中科院分区：
文献类型：
--
作者：
Kachalova, Galina S.;Bourenkov, Gleb P.;Bartunik, Hans D.
烟酸节杆菌氧化降解烟碱的途径包括两种基因和结构上不相关的黄酶，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6-羟基烟碱的L型和d型具有绝对的立体特异性。我们用同构/多波长反常色散相结合的方法在1.95埃分辨率下解算了6HLNO的晶体结构。6HLNO同型二聚体的每个亚基的整体结构和单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，一个二酰基甘油磷脂分子被发现与6HLNO的每个原聚物非共价结合。脂肪酸链占据疏水通道，这些通道深入到每个亚基的底物结合区域的内部。溶剂暴露的甘油磷酸部分位于亚基-亚基界面。我们进一步在2.05埃分辨率下解析了二硫代酸还原6HLNO与天然底物6-羟基- l -尼古丁的配合物的晶体结构。基底在紧密腔中的位置表明，这种非生产性配合物的结合几何形状可能与氧化条件下的结合几何形状非常相似。所观察到的结合底物相对于黄素腺嘌呤二核苷酸辅因子的异alloxazine环的取向适合于氢化物转移脱氢。在形成底物分子手性中心的碳原子上。比较6HLNO和6-羟基- d -尼古丁氧化酶的底物结合模式，基于与d -底物的配合物模型，提出了对这两种酶的立体特异性的解释。这两种酶的作用是使对映体底物相对于黄素平面呈镜像对称。2009爱思唯尔有限公司版权所有。
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 angstrom 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 angstrom 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. (C) 2009 Elsevier Ltd. All rights reserved.