Characterization of conserved active site residues in class I nitronate monooxygenase.
Characterization of conserved active site residues in class I nitronate monooxygenase.
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I 类硝基单加氧酶中保守活性位点残基的表征。
DOI：
10.1016/j.abb.2019.07.023
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发表时间：
2019
影响因子：
3.9
通讯作者：
Gadda,Giovanni
中科院分区：
文献类型：
--
作者：
Su,Dan;Aguillon,Christopher;Gadda,Giovanni
丙酸3-硝基酸（P3N）是一种天然毒素，可不可逆地抑制线粒体琥珀酸脱氢酶。P3N中毒会导致各种神经系统疾病，甚至死亡。铜绿假单胞菌PAO1中的硝酸单加氧酶（NMO）是第一个在细菌中被鉴定的NMO，是一类NMO的典范。在这里，我们假设P3N的羧酸基可能与酶活性位点保守的四个酪氨酸或赖氨酸残基中的一个或多个形成氢键。在野生型酶中，kcat值在pH 6.0 ~ 11.0之间与pH无关，而kcat/ kp3n值在高pH下下降，这表明需要一个pkvalue为9.5的质子化基团来结合阴离子底物。对酶的紫外可见吸收光谱进行pH滴定，发现随着pH的增加，吸光度在297 nm处增加，定义pkk值为9.5，Δε297 nmof为2.4 M−1cm−1，这与酪氨酸对底物结合的重要作用一致。相反，氧化黄素的N3原子没有电离，可能是因为它的pka2受到酶活性部位酪氨酸电离的干扰。Y109F、Y254F、Y299F、Y303F和K307 M取代对酶的稳态动力学参数影响较小（<3.5倍）。在所有突变酶中，kcat/ kp3n值与野生型酶的差异小于2.5倍，这表明没有一个残基是底物结合所必需的。
Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 was the first NMO characterized in bacteria and serves as a paradigm for Class I NMO. Here, we hypothesized that the carboxylate group of P3N might form a hydrogen bond with one or more of the four tyrosine or a lysine residues that are conserved in the active site of the enzyme. In the wild-type enzyme, thekcatvalue was pH independent between pH 6.0 and 11.0, while thekcat/KP3Nvalue decreased at high pH, suggesting that a protonated group with a pKavalue of 9.5 is required for binding the anionic substrate. A pH titration of the UV–visible absorption spectrum of the enzyme showed an increased absorbance at 297 nm with increasing pH, defining a pKavalue of 9.5 and a Δε297 nmof 2.4 M−1cm−1, consistent with a tyrosine being important for substrate binding. The N3 atom of the oxidized flavin, instead, did not ionize likely because its pKawas perturbed by the ionization of a tyrosine in the active site of the enzyme. The Y109F, Y254F, Y299F, Y303F, and K307 M, substitutions had small effects (i.e., <3.5-fold) on the steady-state kinetic parameters of the enzyme. With all mutated enzymes, thekcat/KP3Nvalue was less than 2.5-fold different from the wild-type enzyme, suggesting that none of the residues is solely essential for substrate binding.