Brian G. Fox
Our primary research interests are directed toward understanding the structural and functional properties of the diiron enzymes. The active sites of these enzymes contain two iron atoms bound to the protein backbone by a relatively conserved spacing of histidine and glutamate residues.
Proposed structure for the diiron center in the stearoyl-ACP desaturase. The enzymes containing diiron centers are responsible for a wide variety of important biological reactions, including aspects of aerobic DNA biosynthesis, hydrocarbon and pollutant oxidation, and the formation of unsaturated fatty acids. Our current studies are focused on two different types of enzyme complexes. One is the plant stearoyl acyl carrier protein _9 desaturase (_9D), a soluble enzyme that utilizes O2 and NADPH to catalyze oxidative desaturation. _9D is ultimately responsible for the biosynthesis of oleic acid, the most abundant of the nutritionally and commercially valuable unsaturated fatty acids. The second enzyme complex under study is the bacterial toluene-4-monooxygenase, a four protein enzyme complex that is being used to clean up toxic waste contaminations in soils and ground water. Our hypothesis is that the variation in catalytic properties of the diiron enzymes is a function of the geometry and flexibility of conserved amino acid ligands to the diiron center and of the chemical nature of the amino acids present in the immediate vicinity of the active site. We are working to define these molecular details, and to use this knowledge in conjunction with modern protein engineering strategies to transform the reactivity of the diiron enzymes in new and beneficial ways. Our research group makes extensive use of biochemical, catalytic and spectroscopic techniques as metalloprotein 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 these enzyme complexes. Broadly stated, our 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. It is reasonable to assume that the catalytic diversity of the proteins containing diiron centers is provided by the many possibilities for variation in the ligand types and coordination numbers, by the geometry of ligand binding, and by 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. Stearoyl acyl carrier protein _9 desaturase. In order to provide large amounts of purified enzyme required for our studies, we have developed new fed-batch fermentation methods for overproduction of recombinant metalloproteins using pET vectors in Escherichia coli BL21(DE3) (~1 g of pure _9D per liter of culture medium). The method uses a computer-controlled fermenter housed in the Enzyme Institute, inexpensive lactose in substitution for expensive IPTG as the inducer, and a minimal medium (facilitating 57Fe-enrichment for our collaborative Mšssbauer studies with Prof. E. MŸnck of Carnegie Mellon University, but also suitable for 13C- and 15N-enrichment required for high resolution NMR studies). Relative to standard protocols, we have shown how to obtain a 400-fold decrease in costs, and up to a 100-fold increase in the yield of recombinant proteins. We have proposed this method may be of use to other researchers studying recombinant proteins undergoing post-translational modification, and indeed, many research groups and industrial firms throughout the country have already adopted these methods. We have used optical and resonance Raman spectroscopies (in collaboration with Profs. J. Sanders-Loehr and T. M. Loehr, Oregon Graduate Institute) to prove the presence of an oxo-bridge in the _9D diiron center, and based upon this work and upon primary sequence analyses, we have proposed a structural model for the diiron center ligation (shown above), which has now been confirmed by X-ray studies (Dr. J. Shanklin, Brookhaven National Lab, personal communication). We are continuing these spectroscopic investigations of the the _9D diiron center, and are now performing studies of exogenous ligands such as N3- and CN- as probes of the O2 coordination site required for desaturation catalysis. Toluene-4-monooxygenase. We have recently completed the purification of the diiron center-containing oxygenase, the [2Fe-2S] Rieske center-containing ferredoxin, and the regulatory protein of this enzyme complex. Our results show toluene-4-monooxygenase is an evolutionary combination of the oxygenase and regulatory components from the soluble methane monooxygenase complex, and the ferredoxin and reductase proteins from bacterial aromatic dioxygenase complexes. One consequence of this natural evolutionary process appears to be the rapid appearance of novel enzymes capable of degrading a wide variety of xenobiotic substances in the environment. In collaboration with the lab of Prof. Hazel Holden in the Enzyme Institute, we have crystallized the regulatory protein of the toluene-4-monooxygenase, obtained a complete electron density map at 2.8 A, and have presently found one heavy metal derivative suitable for phase angle determinations. We are continuing our search for other heavy metal derivatives so that the 3-D structure of this novel regulatory protein can be solved. Efforts to crystallize the other proteins of the complex are also under way, as well as 2-D and 3-D NMR studies of the Rieske ferredoxin and regulatory protein in collaboration with the lab of Prof. J. Markley, University of Wisconsin. A further aspect of our research on the toluene oxidation complex has been undertaken as a collaborative venture with the start-up biotechnology company Envirogen, Inc., based in Lawrenceville, NJ. Together, we have used molecular biological methods to perform both random and site-directed mutagenic modifications in the proposed active site of the monooxygenase and have been able to generate new isoforms of the enzyme that have increased rates of oxidation for pollutants and that exhibit changes in substrate specificities for multicomponent hydrocarbon mixtures. These exciting results clearly demonstrate the soundness of our strategy to improve the catalytic versatility of the toluene hydroxylase and other diiron enzymes. Our future research efforts in this area will focus on correlating the observed changes in function of these new isoforms to the structural variations in the diiron enzyme active site using our well-defined combination of biochemical, catalytic, and structural approaches.