W0102

Engineering Regio-Selectivity of Product Formation by Naphthalene Dioxygenase. Daniel J. Ferraro^*#, Adam L. Okerlund^‡, Wendie Dockstader^*, David T.Gibson^§ & S. Ramaswamy^{*‡ *}Dept. of Biochemistry, Univ. of Iowa, ^§Dept. of Microbiology, Univ. of Iowa, ^‡Dept. of Chemical & Biochemical Engineering, Univ. of Iowa, ^#Univ. of Iowa Medical Scientist Training Program.

The addition of dioxygen by the enzyme Naphthalene dioxygenase (NDO) is carried out on a variety of substrates. These reactions are regio, stereo and enantiomerically specific, for example, the dihydroxylation of naphthalene to cis-1R,2S-dihydrodiol. Multiple mutants that altered the active site of the enzyme to favor different regio and stereo selectivity have been identified. Using Biphenyl as a substrate, wild-type NDO favored the production of cis-Biphenyl 2,3-dihydrodiol over cis-Biphenyl 3,4-dihydrodiol in a ratio of 87% to 13%. Using the same substrate, the F352V mutant favored the 3,4 dihydrodiol over the 2,3 dihydrodiol in a ratio of 95.6% to 4.4%. A second mutant, A206I/H295I/V260N, produced over 99% 2,3-dihydrodiol product from the biphenyl substrate. We have been able to overexpress and purify these proteins. In order to understand the structural basis of the Regio-selective product formation, we are currently in the process of structure determination of the mutants. Results of these studies will be presented.