Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase

Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. The diiron center is contained in the tmoA polypeptide of the hydroxylase component [(alpha beta gamma)(2), M-r approximate to 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for pam hydroxylation of toluene (k(cat), approximate to 2 s(-1) with respect to an alpha beta gamma protomer), o-xylene (k(cat) approximate to 0.8 s(-1)), m-xylene (k(cat) approximate to 0.6 s(-1)), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (k(cat) approximate to 0.4 s(-1)), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximate to 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The k(cat) of purified Q141C for toluene oxidation was approximate to 0.2 s(-1). Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relative to the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 and F205 are suggested to lie in a hydrophobic region closer to the Fe-A iron site, while I180 will be closer to Fe-B. These studies reveal that changes in the hydrophobic region predicted to be nearest to Fe-A can influence the regiospecificity observed for toluene 4-monooxygenase.