The Guengerich laboratory is interested in the enzymes involved in the activation and processing of xenobiotic chemicals (i.e., those not normally found in the body, such as drugs and carcinogens).
Mechanisms of Oxidation of Drugs & Carcinogens Catalyzed by Human P450s
(NIH R37 CA90426) involves the cytochrome P450 (P450) enzymes. Work involves the use of site-directed and random mutagenesis and the heterologous expression of human P450 enzymes in bacteria. The P450 enzymes are purified and used in experiments directed at understanding details of catalysis. The work involves chemical synthesis and analysis, spectroscopy, and kinetics. A new focus is on understanding the function of the human “orphan” P450s, and a related aspect deals with the biosynthesis of endogenous mammalian opioids.
Structures and Function of P450 Enzymes in Steroid Hormone Biosynthesis
(NIH GM R01 GM103937) is a collaboration with Prof. M. Egli dealing with two P450 enzymes involved in steroid biosynthesis, P450 17A1 and 21A1. P450 17A1 catalyzes the 17α-hydroxylation of progesterone and pregnenolone and then the subsequent “lyase” reaction to androstenedione or dehydroepiandrosterone. In fish, the P450 17A1 does both reactions but a second protein, P450 17A2, does only the former. We are analyzing the kinetics of the 2-step reaction and also have a crystal structure of zebrafish P450 17A2. P450 21A2 catalyzes the 21-hydroxylation of progesterone and 17α-OH progesterone, and we have a crystal structure of bovine P450 21A2 to use for hypotheses about the structural basis of the >100 clinically known defects in the enzyme. Kinetic analysis of the defective forms of P450 21A2 is also underway.
Biochemistry of P450 Oxidations Relevant to Sodium Transport and Hypertension
(NIH P50 DK038226) is a program project grant, which this work is a part of. P450 enzymes, particularly in the 4A and 2C subfamilies ( and also 2F and 2J) convert arachidonic acid into ω-hydroxy and epoxide derivatives that somehow affect blood pressure. This work is based on that of Prof. Capdevila and involves biochemical studies of these P450s, transgenic mouse models, and regulation of the system through signaling, as well as applications to patients.
Polymerase Interactions with Carcinogen-Modified DNA
(NIH R01 ES10375) involves how polymerases interact with carcinogen-modified DNA to produce base-pair and frameshift mutations. The work focuses on the use of site-specifically modified oligonucleotides (modified with carcinogens) and polymerase enzymology, including prokaryotic models and some mammalian polymerases and involves pre-steady-state and steady-state kinetic analysis of the polymerase reaction cycle, plus the use of X-ray crystallography in defining the relationships of structure and function, in collaboration with Prof. M. Egli.
Bioactivation of Halogenated Hydrocarbons & Bifunctional Electrophiles
(NIH R01 ES10546) involves how activated carcinogens react with proteins and DNA. The focus is on a series of halogenated hydrocarbons (trihaloethylenes, ethylene dibromide, and methylene halides) and other bifunctional electrophiles, e.g. butadiene diepoxide. Some of these chemicals are activated by conjugation with the tripeptide glutathione (GSH) by GSH transferases or a DNA repair protein (O6-alkylguanine transferase). Bacterial genotoxicity systems are utilized, as well as many chemical and spectroscopic approaches.