Pikus J D, Mitchell K H, Studts J M, McClay K, Steffan R J, Fox B G
The Institute for Enzyme Research, Graduate School, Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA.
Biochemistry. 2000 Feb 1;39(4):791-9. doi: 10.1021/bi992187g.
The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O(2)-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallographically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O(2) activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-)(1), respectively, and k(cat)/K(M) values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased approximately 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2, 5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.
来自门多萨假单胞菌KR1的双铁酶甲苯4-单加氧酶催化依赖于NADH和O₂的甲苯羟基化反应。序列比对和光谱研究相结合表明，T4MO的活性位点结构与晶体学表征的甲烷单加氧酶羟化酶密切相关。在甲烷单加氧酶羟化酶中，活性位点残基T213被认为通过与细胞色素P450的某些提议类比来参与O₂的活化。在这项工作中，甲苯4-单加氧酶羟化酶中可比残基T201的诱变已被用于研究活性位点羟基在催化中的作用。基于体内靛蓝形成试验鉴定出了五种保留催化活性的异构体（T201S、T201A、T201G、T201F和T201K），并报道了纯化的T201S、T201A和T201G变体的详细表征。这些异构体的kcat值分别为1.2、1.0和0.6 s⁻¹，kcat/KM值相对于天然异构体仅相差约4倍。此外，这些异构体表现出80 - 90%的偶联效率，这也与天然异构体>94%的偶联效率相当。对于T201S、T201A和T201G异构体，甲苯羟基化的区域特异性与天然异构体几乎相同，对甲酚占总产物分布的90 - 95%。相比之下，T201F异构体导致产物分布发生显著变化，邻甲酚和对甲酚的比例为1:1。此外，T201F异构体使苯甲醇的量增加了约10倍。对于与对二甲苯的反应，先前的研究表明天然异构体反应生成4-甲基苄醇和2,5-二甲基苯酚的比例为4:1 [皮库斯，J. D.，斯塔茨，J. M.，麦克雷，K.，斯特凡，R. J.，和福克斯，B. G.（1997年）《生物化学》36，9283 - 9289]。作为比较，T201S、T201A和T201F异构体给出了这些产物稍宽松的3:1比例，而T201G异构体给出了显著宽松的1:
