Koetter Jochen W A, Schulz Georg E
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, 79104 Freiburg im Breisgau, Germany.
J Mol Biol. 2005 Sep 16;352(2):418-28. doi: 10.1016/j.jmb.2005.07.041.
The crystal structure of 6-hydroxy-d-nicotine oxidase (EC 1.5.3.6) was solved by X-ray diffraction analysis in three crystal forms at resolutions up to 1.9 A. The enzyme is monomeric in solution and also in the mother liquor but formed disulfide-dimers in all crystals. It belongs to the p-cresol methylhydroxylase-vanillyl-alcohol oxidase family and contains an FAD covalently bound to the polypeptide. The covalent bond of this enzyme was the first for which a purely autocatalytic formation had been shown. In contrast to previous reports, the bond does not involve N(epsilon2) (N3) of His72 but the N(delta1) (N1) atom. The geometry of this reaction is proposed and the autoflavinylation is discussed in the light of homologous structures. The enzyme is specific for 6-hydroxy-D-nicotine and is inhibited by the L-enantiomer. This observation was verified by modeling enzyme-substrate and enzyme-inhibitor complexes, which also showed the geometry of the catalyzed reaction. The binding models indicated that the deprotonation of the substrate rather than the hydride transfer is the specificity-determining step. The functionally closely related 6-hydroxy-L-nicotine oxidase processing the L-enantiomer is sequence-related to the greater glutathione reductase family with quite a different chainfold. A model of this "sister enzyme" derived from known homologous structures suggests that the reported L-substrate specificity and D-enantiomer inhibition are also determined by the location of the deprotonating base.
通过X射线衍射分析，以三种晶体形式解析了6-羟基-D-尼古丁氧化酶（EC 1.5.3.6）的晶体结构，分辨率高达1.9埃。该酶在溶液和母液中均为单体，但在所有晶体中形成二硫键二聚体。它属于对甲酚甲基羟化酶-香草醇氧化酶家族，含有一个与多肽共价结合的FAD。这种酶的共价键是首个被证明为纯自催化形成的共价键。与先前的报道不同，该键不涉及His72的N(ε2)（N3），而是N(δ1)（N1）原子。提出了该反应的几何结构，并根据同源结构讨论了自黄素化作用。该酶对6-羟基-D-尼古丁具有特异性，并受到L-对映体的抑制。通过对酶-底物和酶-抑制剂复合物进行建模验证了这一观察结果，该模型还展示了催化反应的几何结构。结合模型表明，底物的去质子化而非氢化物转移是决定特异性的步骤。功能密切相关的处理L-对映体的6-羟基-L-尼古丁氧化酶与更大的谷胱甘肽还原酶家族存在序列相关性，其链折叠方式截然不同。基于已知同源结构推导的这种“姐妹酶”模型表明，所报道的L-底物特异性和D-对映体抑制作用同样由去质子化碱基的位置决定。
