Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1)).
Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1)).
复制标题
来自嗜甲基菌（sp. W(3)A(1)）的重组野生型和三甲胺脱氢酶 C30A 突变体的结构和生化特征。
DOI：
10.1021/bi9927181
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Mathews,FS
中科院分区：
文献类型：
--
作者：
Trickey,P;Basran,J;Lian,LY;Chen,Z;Barton,JD;Sutcliffe,MJ;Scrutton,NS;Mathews,FS
三甲基胺脱氢酶（TMADH）是一种铁硫黄蛋白，可催化三甲基胺的氧化脱甲基作用形成二甲胺和甲醛。它含有一种独特的黄素，以6-S-半胱氨酰FMN的形式存在，沿着黄素异咯嗪环的N5−N10轴沿着弯曲约25°。这种不寻常的构象被认为是调节黄素的性质，以促进催化，并被假定为在黄素C6原子上共价连接到Cys-30的结果。我们在这里报告的晶体结构的重组野生型和C30 A突变TMADH酶，都确定在2.2 μ m分辨率。结合晶体学和NMR研究揭示了无机磷酸盐的存在下，在FMN的结合位点的deflavo部分的重组野生型和C30 A蛋白。重组酶中紧密结合的无机磷酸盐的存在解释了不能重建体内产生的重组野生型和C30 A酶的去鞭毛形式。C30 A TMADH中的活性位点结构和黄素构象与重组和天然TMADH中的活性位点结构和黄素构象相同，因此揭示了与预期相反，6-S-半胱氨酰FMN连接并不负责TMADH中黄素沿N5−N10轴沿着25°蝶形弯曲。计算量子化学研究强烈支持蝴蝶弯曲在调节黄素的氧化还原性质中的作用。溶液研究揭示了野生型和C30 A蛋白的动力学行为的主要差异。计算研究揭示了Cys-30的Sγ原子对底物结合的迄今未被认识的贡献，以及Cys-30在底物与酶活性位点中的6-S-半胱氨酰FMN的最佳几何对齐中的作用。
Trimethylamine dehydrogenase (TMADH) is an iron−sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by ∼25° along the N5−N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 Å resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25° butterfly bending along the N5−N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the Sγatom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.