Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not requiredfor catalysis

The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 ca talyzes 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 crystallo-graphically charact erized methane monooxygenase hydroxylase. In the methane monooxygenase hydr oxylase, active site residue T213 has been proposed to participate in O-2 a ctivation 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, an d T201K) that retain catalytic activity based on an in vivo indigo formatio n assay were identified, and detailed characterizations of the purified T20 1S, T201A, and T201G variants are reported. These isoforms have k(cat) valu es 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. Moreo ver, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isofor m. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluen e 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 similar to 10-fold with the T201F isoform. For reacti on with p-xylene, previous studies have shown that the native isoform react ed to give 4-methybenzyl alcohol and 2,5-dimethylphenol in a 4:1 ratio [Pik us, 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 i soforms gave a slightly relaxed 3:1 ratio of these products, while the T201 G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these stud ies, we conclude that the hydroxyl group of T201 is not essential to mainta ining the turnover rate or the coupling of the toluene 4-monooxygenase comp lex. However, changing the volume occupied by the side chain at the positio n of T201 can lead to alterations in the regiospecificity of the hydroxylat ion, presumably by producing different orientations for substrate binding d uring catalysis.