Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity.
Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an approximately 20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents approximately 60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absence or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V(max)/K(M) specificities differing by approximately 2-4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.
Mitchell
Kevin H
KH
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Studts
Joey M
JM
Fox
Brian G
BG
D001665:Binding Sites; D002384:Catalysis; D004579:Electron Transport; D007527:Isoenzymes / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008968:Molecular Conformation; D016296:Mutagenesis; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism*
D007527:Isoenzymes; D014050:Toluene; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C114360:toluene 2-xylene monooxygenase