Threonine 201 in the Diiron Enzyme Toluene 4-Monooxygenase Is Not Required for Catalysis

Abstract

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.

Structural consequences of effector protein complex formation in a diiron hydroxylase

“…A direct transfer of a proton to a peroxo intermediate or a positioning of water for this transfer have been proposed for the conserved Thr residue (27), and the participation of Thr-201 in orientation of HOH5 is consistent with the latter role in T4moH. Because mutagenesis showed that Thr-201 was not essential for steady-state catalysis (28), this residue may also participate in other steps in the reaction cycle, such as stabilization of the rearranged conformation for electron transfer.…”

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

“…The rotameric conformation of this Asn also changes with the active site oxidation state; it points inward toward the diiron center in the mixed-valent and reduced forms and away from it when the iron atoms are oxidized ( Figure 5B) (48,49). Mutagenesis studies on TMOs suggest that the Thr is not essential for catalysis under steady state conditions (60), whereas the Asn is important for turnover and protein component interactions. 5 The positions of this pair of Thr and Asn residues in both protomers from the native and SeMet PHH structures differ significantly from what has been observed for oxidized, reduced, and mixed-valent MMOH (8,46,48,49) as well as for oxidized and Mn 2+ -substituted ToMOH (9,57).…”

Protein Engineering of Toluene- o -Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

“…strain JS150 have been predicted by Pikus et al (25). The T201 residue encoded by tmoA in T4MO has been studied by saturation mutagenesis, and T4MO Q141C, Q141V, I180F, and F205I mutants with mutations in tmoA have been studied previously by using site-directed mutagenesis (24,25). For oxidation of m-xylene by the Q141C T4MO mutant, 3-methylbenzyl alcohol formation increased sixfold from 2.2% to 11.7%, and for p-xylene oxidation the product distribution completely switched to 2,5-dimethylphenol (78%) from 4-methylbenzyl alcohol (22%).…”