Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity.
Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity.
复制标题
羟化酶活性位点残基和效应蛋白结合联合参与甲苯 4-单加氧酶区域特异性的对位至邻位调节。
DOI：
10.1021/bi012036p
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Fox,BrianG
中科院分区：
文献类型：
--
作者：
Mitchell,KevinH;Studts,JoeyM;Fox,BrianG
甲苯4-单加氧酶（T4 MO）是一种具有高度区域专一性的二铁羟化酶。这种保真度为评估活性位点残基与蛋白质复合物形成之间的相互作用提供了基础，以产生重要的生物学结果。T4 MO催化复合物（羟化酶、T4 moH和效应蛋白T4 moD）的功能在效应蛋白浓度、T4 MO电子转移组分（Rieske铁氧还蛋白、T4 moC和NADH氧化还原酶）的存在以及具有不同羟化区域特异性的突变T4 moH同种型的使用方面进行评价。稳态动力学分析表明，T4 moC和T4 moD与T4 moH形成具有相似亲和力的复合物。在低T4 moD浓度下，稳态羟基化速率线性依赖于T4 moD − T4 moH复合物的形成，而区域特异性和NADH消耗与羟基化之间的耦合效率与T4 moD − T4 moH复合物的内在性质相关。优化的络合物提供了有效的偶联和高区域特异性，对甲酚占甲苯总产物的>96%。用T3 buV（来自甲苯3-单加氧酶的效应蛋白）获得对羟基化的类似偶联和区域特异性，表明效应蛋白结合并不唯一地确定或改变甲苯羟基化的区域特异性。T4 moD的缺失导致羟基化速率降低约20倍，几乎完全解偶联，并且区域特异性降低，使得对甲酚占总产物的约60%。在替代底物的氧化中观察到类似的区域特异性变化，或者在从甲苯氧化中部分去除T4 moD或T3 buV后观察到。研究的突变T4 moH亚型具有明显的Vmax/Km特异性，差异为1.2 - 4倍，偶联效率为88%-95%，表明具有可比的催化功能，但对所有测试底物也表现出独特的区域特异性模式，表明活性位点内独特的底物结合偏好。G103 L异构体具有增强的邻羟基化选择性，除了硝基苯，其产生邻硝基苯酚。G103 L同种型的区域特异性与从甲苯/苯/邻二甲苯单加氧酶亚家族的天然存在的变体中观察到的区域特异性相当。进化和机械的影响，这些研究结果被认为是。
Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity forparahydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD−T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD−T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity withp-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity forparahydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an ∼20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so thatp-cresol represents ∼60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absense or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparentVmax/KMspecificities differing by ∼2−4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity fororthohydroxylation with all substrates tested except nitrobenzene, which gives onlym-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.