Biochemical, catalytic, and spectroscopic studies of redox active enzymes; protein engineering
We have recently established that catalytically essential diiron centers are found in the plant stearoyl-acyl carrier protein Delta 9 desaturase (Delta 9D) and the bacterial toluene-4-monooxygenase (T4MO). These soluble, multicomponent enzymes utilize dioxygen and NADPH to catalyze the oxidation of hydrocarbons. Delta 9D is ultimately responsible for the biosynthesis of oleic acid, the most abundant unsaturated fatty acid, while T4MO catalyzes the para-hydroxylation of toluene. These enzyme complexes are of interest because they can oxidize stable C-H bonds. In addition, we study flavoenzymes that initiate the bacterial utilization of explosive compounds such as nitroglycerin and TNT as nitrogen sources for growth. For all of these enzymes, we are interested in determining the molecular details of the catalytic reactions.
Broadly stated, our research goals are to define the structure and the reactivity of the active site diiron center, to probe the catalytic contributions of the active site protein residues, and to determine the consequences of protein-protein and protein-substrate interactions on the outcomes of enzymic catalysis. Our research group makes extensive use of biochemical, catalytic, and spectroscopic techniques as metalloenzyme active site probes. Through application of these techniques, resting states as well as highly reactive intermediates of the diiron enzyme catalytic cycle, are being characterized. In addition to providing fundamental mechanistic and structural information, these characterizations form the basis for ongoing site-directed mutagenic manipulations of the protein- and substrate-components of the enzyme complex. Since we obtain both of Delta 9D and T4MO from recombinant overexpression systems, we also remain interested in innovative ways to use advanced fermentation technologies to further improve the productivity and yield of our enzymes from these vectors.
It is reasonable to assume that the catalytic diversity of the enzymes containing diiron centers is related to the many possibilities for variation in the ligand types and coordination numbers, in the geometry of ligand binding, and in the polarity of the environment surrounding the diiron center. Highly specific protein-protein interactions must also contribute to the rates and yield of catalytic turnover. Through the detailed characterization of the attributes of these versatile catalysts, we would ultimately like to assemble bioengineered diiron enzymes capable of the oxidative biotransformation of a wide variety of hydrocarbons.
For the flavoenzymes, we are using a coordinated bacteriological, biochemical, engineering, and structural approach to address the problem of remediation of explosive compounds. This collaborative effort involves studies from basic research to field application.