Abstract - Molecular Biochemistry Brian G. Fox (University of Wisconsin) MCB 97-33734 CAREER: Protein Engineering of a Recombinant Diiron Hydroxylase via Mutagenesis and in vivo Selection to Target the Creation of New Catalytic Properties. 1. Technical This study examines a recombinant form of bacterial toluene 4-monooxygenase (T4MO), which is a four protein complex consisting of an FAD and 2Fe-2S containing NADH oxidoreductase (tmoF, 36 kDa); a Rieske ferredoxin (tmoC, 12.3 kDa); a catalytic effector (tmoD, 11.6 kDa); and a diiron hydroxylase (T4MOH, 212 kDa, dimeric quaternary structure composed of the tmoA, tmoB, and tmoE gene products). The objectives are to create, select, and characterize new isoforms of the T4MO complex by bioengineering the diiron active site, which is contained entirely within the tmoA gene. A combination of targeted and random mutagenesis coupled with selection through in vivo complementation is used to alter the regiospecificity of aromatic oxidation in a targeted manner, to oxidize entirely new classes of hydrocarbon substrates, to produce new variations of catalytic reactivity, or to enhance catalyst stability. The T4MO complex offers many unique opportunities to study diiron enzyme catalysis, to investigate the role of protein-protein interactions in catalysis, and to study the energetics of electron transfer between the Rieske ferredoxin and diiron redox centers. It oxidizes many aromatic, aliphatic, heteronuclear, polynuclear, and halogenated hydrocarbons. Because hydrocarbons are the preeminent chemical feedstocks and about 60% of all industrial transformations involve some form of oxidation or reduction, this study is relevant to environment and biotechnology. This research is integrated into discovery-based undergraduate teaching, involving state-of-the-art molecular biology/biochemistry techniques. 2. Non-technical Bacterial toluene 4-monooxygenase (T4MO) is a four protein complex containing two iron atoms in the catalytic active site. Many key biochemical principles are inherent in the function of this multiprotein complex, including the role of metal ions in catalysis, protein interactions, electron transfer processes, substrate specificity, and others. This enzyme oxidizes many aromatic, aliphatic, and halogenated hydrocarbons. Hydrocarbon oxidation is an important topic because hydrocarbons are the preeminent chemical feedstocks and about 60% of all industrial transformations involve some form of oxidation or reduction. This study is aimed to create, select, and characterize new and improved isoforms of T4MO through bioengineering, using a variety of biochemical, catalytic, and physical approaches. This study is integrated also into discovery-based undergraduate teaching, involving state-of-the-art techniques such as DNA isolation and sequencing, expression vector construction, protein expression, protein purification, and structure/ function characterizations. The objective is to contribute to the utility of T4MO complexes as biocatalysts, gaining significant knowledge of their function, and providing a stimulating focus for teaching the theory and practical application of modern biochemical techniques.
