W0188

High Resolution Structures of THT-Dioxygenase Mutants: Insight into Extradiol Catalysis. Magdalini Vamvouka and Andrew Mesecar, The Center for Pharmaceutical Biotechnology and the Dept. of Medicinal Chemistry and Pharmacognosy, The Univ. of Illinois at Chicago, Chicago, IL 60607.

The oxidative cleavage of ortho-dihydroxy aromatic compounds by extradiol and intradiol dioxygenases is essential in microbial degradation of naturally occurring and man-made aromatic molecules.

2,4,5-Trihydroxytoluene dioxygenase (THT-DO) is the terminal enzyme in the pathway for biodegradation of 2,4-dinitrotoluene in Burkholderia sp. strain DNT. It utilizes an Fe (II) center as a cofactor and O₂ as a second substrate, and it catalyzes the extradiol cleavage of 2,4,5-trixydroxytoluene. Extradiol catalysis commonly proceeds via an ordered kinetic mechanism whereby a complex between Fe (II) and the aromatic substrate is formed first, followed by binding of O₂ to the iron. Electron rearrangement in the active site yields a bridged peroxy adduct which then undergoes a Crieege rearrangement to yield a lactone intermediate. Hydrolysis of the lactone then produces the ring opened product. Our high-resolution crystal structures of native THT-DO and the enzyme in complex with the inhibitor 4-nitrocatechol, suggest that H206, located near the proposed O₂ binding site, may be the proton donor for O-O bond cleavage of the peroxy intermediate species, and that Y261, which is 3 Å from the Fe atom, may act as a base in the reaction via a proton shuttle with H252 . To elucidate the mechanism of extradiol catalysis, we constructed three point mutants of THT-DO: Y261F, H252A and H206A. We have crystallized and determined the high-resolution crystal structures of apo-H252A (1.4 Å, R_crys=19.3%, R_free=20.7%), apo-Y261F (1.37 Å, R_crys=19.2%, R_free=21.0%), and the 4-nitrocatechol bound form of Y261F (1.2 Å, R_crys=20.4%, R_free=21.7%). Preliminary kinetic data of wild-type, H252A and Y261F THT-DO indicate significant reduction of the enzymatic activity upon mutation. We will discuss correlations between the kinetic and structural differences of these mutant enzymes. This research is supported by the Office of Naval Research Awards N000140210956.