Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Liquid state NMR spectroscopy as a tool to obtain structural and binding information of drug molecules metabolized by P450BM3 enzymes
Cytochrome P450 is a heme protein family ubiquitous in all domains of life and involved in many biological processes, including the degradation and activation of drugs by catalyzing the monooxygenation of a ligand (1). Cytochrome P450BM3 from Bacillus megaterium is one of the most promising monoxygenases for biotechnological applications because it is the most active P450 so far identified. Mutated forms of P450BM3 are highly efficient biocatalysts to metabolize steroids and other drugs in-vitro in order to produce potential new lead compounds (2). In this contribution, liquid state NMR spectroscopy is shown to be a valuable tool to provide a structural basis for drug metabolism by P450BM3 enzymes. First, the regioselective hydroxylation of steroids by P450BM3 enzymes will be discussed. Secondly, a new methodology for acquiring homonuclear decoupled proton NMR spectra of drug metabolites will be discussed.
We show that by a single mutation (A82W) in the substrate binding pocket of P450BM3 mutants, the regioselective hydroxylation of steroids at position 16-ß is largely increased. Moreover, this enhanced regioselective hydroxylation is further investigated by means of binding affinity, as studied by UV-VIS spectroscopy, and the orientation of the steroid testosterone in the heme-active sites of these mutants, as studied by T1 paramagnetic relaxation NMR. It is shown that testosterone has an increased binding affinity and preferred orientation in the A82W mutant when compared to its wild-type counterpart.
NMR spectroscopy is not only a very powerful tool to provide information on ligand binding, but its main power is to provide structural information on large molecules, like RNA’s, DNA’s and enzymes, and small molecules. The determination of structures of small molecules, i.e. metabolites which are formed by P450BM3 enzymes, by liquid state NMR is a standardized but complicated procedure (2). Small molecules often have a dense network of J-coupled protons, which hampers the assignment of NMR spectra, due to broad multiplets and subsequent spectral overlap. We will show a novel methodology to acquire homonuclear decoupled proton NMR spectra with a high resolution (~1Hz) and higher sensitivity than currently reported for these types of NMR experiments.
1 Department of Biophysical Chemistry, Radboud University Nijmegen