title: Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone.
abstract: Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 +/- 0.9 to 10.7 +/- 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 +/- 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 +/- 0.2 versus 0.21 +/- 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
authors.affiliation: Department of Chemical Engineering, University of Connecticut, Storrs, CT 06269-3222, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tao
authors.forename: Ayelet
authors.initials: A
authors.lastname: Fishman
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D019943:Amino Acid Substitution; D001554:Benzene / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002396:Catechols / Q000378:metabolism*; D003408:Cresols / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019800:Phenol / Q000378:metabolism; D021122:Protein Subunits; D044203:Pseudomonas mendocina / Q000201:enzymology* / Q000235:genetics; D011748:Pyrogallol / Q000378:metabolism; D021281:Quantitative Structure-Activity Relationship; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism
chemical_list: D002396:Catechols; D003408:Cresols; D006873:Hydroquinones; D021122:Protein Subunits; D011748:Pyrogallol; C032538:4-cresol; D019800:Phenol; D014050:Toluene; D010105:Oxygenases; C068610:toluene-4-monooxygenase; C066344:3-methoxycatechol; D001554:Benzene; C034221:catechol