Performing Department
Biochemistry
Non Technical Summary
Biosynthetic strategies for hydrocarbon production from fatty acid metabolites provide a means to expand on a depleting petrochemical supply. Recently identified enzymes for alkane and alkene synthesis utilize iron-containing cofactors that catalyze highly atypical, and in many cases cryptic, carbon-carbon scission reactions. As a result, their mechanism and biosynthetic potential has remained largely untapped. We proposed to study two newly discovered enzymes that have the potential to both produce drop-in fuel replacements and are also important in the proliferation of microbial pathogens and insect development, termed UndA and CYP4G, respectively. Both enzymes differentiate from previously studied hydrocarbon-forming enzymes in our laboratory through their use of atmospheric dioxygen, rather than the toxic reactive oxygen species hydrogen peroxide, as a co-substrate. However, the mechanisms of both remain poorly understood. Coordinated biophysical, structural and mechanistic studies of UndA and CYP4G will provide important parallels to other mechanisms of hydrocarbon biosynthesis, aid in understanding the basis for reaction diversity catalyzed by iron-dependent enzymes, andaprovide new avenues to enable inhibitor design.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
My research program globally focuses on understanding and leveraging enzymes that are involved in the synthesis and metabolism of natural products of industrial and biomedical value. One very area of active investigation is the biochemical characterization of enzymes involved in transforming fatty acid metabolites into hydrocarbons that can serve as fuels and important chemical building-blocks. Although phylogenetically diverse species have long been recognized to produce hydrocarbons for a variety of important physiological roles, most of the enzymes responsible have only been identified within the past decade and their mechanisms remain enigmatic. One of the most intriguing chemical aspects of these enzymatic conversions is the use of iron-containing cofactors that are more commonly known to catalyze oxygen insertion reactions for C-C cleavage. It is our ultimate goal is to understand how specific structural aspects are coordinated with the chemistry of oxygen-activation to afford very different reaction outcomes. This work focuses on elucidating the biochemical basis for O2-dependent alkene production by the non-heme enzyme UndA and alkane formation by the cytochrome P450 CYP4G1.Objective 1: Characterize the cofactor and mechanism of UndA and relate its activity to the H2O2-dependent heme decarboxylase cytochrome P450 OleTObjective 2: Characterize the biochemical mechanism of CYP4G1
Project Methods
Objective 1: Characterize the cofactor and mechanism of UndABinding and metabolism studies: An appraisal of the panel of substrates that UndA can accept and metabolize will be informative for providing evidence for the intermediacy of a high-valent intermediate and pinpointing points of contact between the protein and substrate that are required for reinforcing C-C cleavage. Substrate binding will be monitored using isothermal calorimetry (ITC) or through the use of fluorescent (dansyl) or EPR-active (doxyl) fatty acid analogs. This approach has been very useful for (a) identifying and/or modifying sites of interaction between the substrate and protein (b) monitoring dynamic changes during catalysis, and (c) appraising the degree of substrate mobility along the acyl chain. The extracted KD values (and thermodynamic parameters) will be used to guide transient and spectroscopic studies. For turnover studies, we have found several chemical and biological redox systems support high levels of undecene formation. This facilitates our detection of minor side-products using the GC-MS methods. We will first test the CL specificity of saturated FAs, ranging from C24 to C4 to test for the presence of hydroxylated products (even minor) in the metabolism of non-native substrates. If we see alcohol products, we will test for the integration of oxygen which would verify involvement of a high-valent species. We will generate a series of site-directed mutations along the predicted substrate-binding channel (e.g. Trp191, Val65 and Tyr58) of UndA to test for their influence on activity and chemoselection.Spectroscopic Studies: Our initial characterization of the UndA:C12 FA complex by Mössbauer indicates that at least one Fe site is perturbed upon the addition of substrate in the diferrous state. We will perform additional spectroscopic investigations into the UndA:substrate complexes that we find support catalysis. We will utilize EPR of the mixed-valent (MV) state as an initial probe. Given the asymmetric nature of the cluster, the reduction potentials of UndA should be sufficiently separated to access this state. Direct coordination of an FA would be expected to result in a pronounced perturbation of the EPR spectrum and alter the degree of antiferromagnetic coupling. We will also extend our studies to other spectroscopies such as resonance Raman and x-ray absorption spectroscopy (XAS) for more detailed structural characterization of the cluster.Crystallographic Studies: A main target is to solve the crystal structure of UndA with the appropriate di-Fe cofactor intact with and without bound substrates. We have reproduced the Pp-UndA crystallization conditions used by the Joint Center for Structural Genomics (PDB: 3OQL), have collected datasets at the APS synchrotron, and solved preliminary structures of both the apo- and mono-Fe loaded enzymes at a resolution of 2.3 Å or better. Based on this, we anticipate that there is a high probability that the dinuclear form of the enzyme will also crystallize with this condition. However, if it does not, we will screen others using high-throughput (HT) methods offered from in house or external screening resources.Transient kinetics: Our GC studies show that the single-turnover conditions we have developed for UndA are catalytically competent and have revealed at least one intermediate (λmax ~ 560 nm) that can be formed at high levels. We will extend this approach to more inert substrate analogs to trap additional intermediatesand clarify the active-oxidant involved.Objective 2: Characterize the biochemical mechanism of cytochrome P450 CYP4G1Reconstitution of CYP4G1 activity: Elucidating the mechanism of CYP4G will help to assess its biotechnological potential and serve as a guide for the preparation of CYP4G inhibitors in future work. As with many eukaryotic P450s, CYP4G1 is embedded in the membrane through a single N-terminal transmembrane helix. Poor expression, and the hydrophobic nature of the protein, has limited studies to heterogenous preparations obtained from detergent-solubilized microsomes and prohibited detailed studies. Wehave overcome this limitation by screening a panel of over 20 different genetic constructs and isolated several that produce milligram quantities of protein in E. coli (per L of culture). Using differential centrifugation, we can isolate, detergent solubilize, and purifythe protein from the membrane fraction. The protein is highly homogeneous and produces the UV/Vis spectra that are diagnostic for functional P450s. As an alternative, we will also pursue expression protocols using baculovirus infected Sf9 cells. It is ideal to study CYP4G1 in its physiologically relevant native bilayer environment, particularly due to the hydrophobic nature of the aldehydic substrates that it metabolizes. We will next assemble CYP4G1 into Nanodiscs which provide a native phospholipid bilayer environment of the enzyme and enable functional studies.Identify the Mechanism for CYP4G1 C-C Scission: Past studies have shown that CYP4G1 metabolizes various CL aldehydes to alkanes and CO2. This reaction is a highly unusual because it involves the integration of oxygen into the one-carbon co-product. However, CO2 is not the expected one-carbon co-product (typically formate) that would result from a deformylation reaction. We will first verify the co-product and identify the source of the integrated oxygen (O2 or H2O) into the one-carbon co-product using 18O2 and MS. As with UndA, we will test whether the metabolism of different CL aldehydes produce alcohols that are indicative of an Fe4+ species. In OleT (and most P450s) the active reagent for CYP4G metabolism is usually the iron(IV)-oxo. However, this type of intermediate would be expected to normally produce a FA from an aldehyde substrate. C-C scission by a peroxo-anion (the likely alternative) is possible but would typically result in a formate co-product. We will use several different strategies to clarify the active species used by CYP4G. Oxygen donors (e.g. organic peroxides) that can form the form the iron(IV)-oxo (but not peroxo) can be used. Likewise, we will perform mutagenesis of active site amino acids that are critical for proton-assisted formation of the iron-oxo in other P450s and are present in CYP4G proteins. If a peroxo- species is the intermediate responsible for CYP4G metabolism, the activity will be unchanged (and possibly enhanced) upon mutation. Similarly, performing the reaction in D2O, which depletes the iron-oxo species due to a kinetic solvent isotope effect, serves as an alternative probe. Finally, the reaction coordinate can be directly followed through the delivery of electrons by gamma-irradiation at low temperatures. This cryoradiolytic methodology has been used by the PI in past studies of heme enzymes and allows one to directly produce iron-peroxide species, and slowly monitor their progression through controlled thermal annealing and EPR.Comparative Crystallographic and Functional Studies: A long-term goal will be to structurally characterize CYP4G1 to determine the origins for the unique reaction coordinate. We will perform sparse matrix screening on the best expressing and functional proteins identified previously and then proceed to fine grid screens using resources available in house (METRIC). As has been demonstrated with other CYPs, the inclusion of substrate(s) and heme-ligands in the mother liquor will be used as a general stratagem to enable crystallization and to determine substrate/inhibitor binding modes. Intriguingly, the predicted active-site construction of CYP4G1 from homology modeling highly resembles that of rat CYP4B1 which has been previously crystallized. We will perform parallel metabolism studies and solve structures of CYP4B1 with aldehydes bound to determine how the two proteins differ with respect to substrate-binding and metabolic profile and guide further mutagenesis studies.