Our laboratory uses x-ray crystallography as a central tool for studying protein structure and mechanism. Historically, crystallography has been a powerful technique for determining protein folds. Now, with the advances in molecular biology, synchrotron radiation, phasing techniques, and computing, crystallography enables researchers to go beyond a single picture of one enzyme from one organism: it is now possible to elucidate structures of entire enzyme pathways and to capture multiple "snapshots" of enzymes as they proceed through their reaction cycles. Our laboratory combines molecular biology, biochemistry, and spectroscopy with state-of-the-art crystallographic techniques to investigate protein function.
Our research focuses on metalloproteins, since the combination of metals with amino acids allows for the unique reactivity that is essential for all living things. The most fundamental of cellular reactions involve metalloproteins, such as carbon fixation, nucleotide biosynthesis, and photosynthesis. Metalloproteins are also vital for the biosynthesis of natural products, catalyzing some of the more complicated steps in biosynthetic pathways. Below are descriptions of selected projects representative of the types of systems we study, the kinds of information we obtain, and our future directions.
Crystallographic Studies of a Family: Ribonucleotide Reductases and Nucleotide Biosynthesis
Ribonucleotide reductases (RNRs) use metallocofactors and radical-based chemistry to catalyze an essential step in DNA biosynthesis, the conversion of ribonucleotides to deoxyribonucleotides, making them excellent antitumor and antiviral drug targets. Three discrete cofactors are used by RNRs: di-iron tyrosyl radical (class I), adenosylcobalamin (class II), and glycyl radical (class III). We are studying all three classes, since each RNR class allows us to probe distinct biochemical questions. We are working to characterize human class I RNR structurally, since it is an important drug target. The first structure of a class II RNR, solved in our laboratory, shows that class II RNRs are ideal for investigating the allosteric regulation of substrate specificity and radical generation. Our goal for class III RNR is to understand how the activase docks to the catalytic subunit for glycyl radical generation. Since each class of RNR offers different challenges and rewards, our studies of RNR now involve obtaining snapshots of the entire RNR family.
Mechanistic Crystallography with Mononuclear Iron Proteins
How do proteins with the same fold and metal catalyze a diverse set of reactions? Mononuclear iron proteins catalyze reactions ranging from hydroxylation to epoxidation to halogenation, and these enzymes function in pathways as diverse as DNA demethylation and antibiotic biosynthesis. Our studies of nonheme iron halogenases reveal how the same protein scaffold can be co-opted to convert an enzyme that hydroxylates to one that halogenates. We discovered a novel coordination environment for the catalytic iron, and the presence of a naturally occurring iron-chloride bond. The active-site architecture suggests a mechanism by which nature can harness the catalytic prowess to perform the most chemically challenging of halogenation reactions. Halogenase chemistry is fascinating in general, and with more than 4,000 halogenated natural products, understanding how these tailoring reactions are catalyzed is of biological and industrial significance. We have solved the first two structures of enzymes in this family: syringomycin E biosynthetic enzyme SyrB2 and putative cytotrienin A biosynthetic enzyme CytC3. Syringomycin is an antifungal agent; cytotrienin A can induce apoptosis.
Capturing Snapshots of AdoMet Radical Proteins
The diversity of radical enzyme reactivity allows for the production of biomolecules and the maintenance of cellular processes that are essential for life. Examples of the remarkable reactions that occur through radical chemistry include those catalyzed by five AdoMet radical proteins: biotin synthase (BioB) and lipoyl-ACP synthase (LipA) convert unactivated C-H bonds into C-S bonds to form the vitamins biotin and lipoate, respectively; pyruvate formate-lyase–activating enzyme (PFL-AE) and class III RNR activase form a glycyl radical species on their target enzymes; and spore photoproduct lyase repairs UV-induced DNA damage. To probe this unusual chemistry, we are investigating the structure and mechanism of numerous AdoMet radical family members. Some of these proteins, such as biotin synthase, have a slow rate of turnover (min–1 to hr–1), making them ideal for crystallographic studies of enzyme intermediates. Using a combination of crystallography and single-crystal spectroscopy, we can follow a series of spectroscopic signals with a microspectrophotometer, allowing for the rapid cryocooling of the crystal at sequential time points along the reaction coordinate.
Structural Elucidation of an Enzymatic Pathway: Biosynthesis of the Antitumor Compound Rebeccamycin
Rebeccamycin is a natural product isolated from Lechevalieria aerocolonigenes and is a prototype for a class of compounds that bind to DNA-topoisomerase I complexes, preventing the replication of DNA and thereby acting as antitumor compounds. Rebeccamycin is synthesized by the action of eight enzymes, with the overall conversion of L-tryptophan, chloride, molecular oxygen, glucose, and a methyl group to the glycosylated indolocarbazole rebeccamycin. We are pursuing the structural characterization of this pathway, which includes a cornucopia of cofactor- and metal-dependent enzymes. Currently, we have solved the structure of the flavin-dependent halogenase RebH and of the flavin-dependent protein RebC. The latter is part of a two-enzyme system (RebP/RebC) that is involved in the conversion of chlorinated chromopyrrolic acid to the rebeccamycin aglycone; this is achieved by an eight-electron oxidation, the mechanism of which is not established. Structural analysis of RebH has allowed us to compare and contrast halogenation reactions catalyzed by a flavin cofactor versus those catalyzed by mononuclear iron centers.
Capturing Snapshots of Metalloproteins in Action: The Regulation of a Transcription Factor by Nickel
Metal ions are essential nutrients for all cells, but their intracellular concentrations must be tightly regulated to avoid toxicity. Nickel ions are particularly important to the physiology of microorganisms such as the model prokaryote Escherichia coli and the human gastric pathogen Helicobacter pylori. In these organisms, incorporation of nickel into enzymes such as [NiFe]-hydrogenase and urease is necessary for metabolic adaptation to changing environmental conditions. In E. coli, Ni concentrations are controlled by transcriptional repression of the Ni-specific ABC transporter. In the presence of excess intracellular Ni, the Ni-dependent repressor NikR binds to an operator sequence, turning off expression of the transporter and decreasing Ni import. In H. pylori, which requires the enzyme urease to survive and colonize the acidic gastric mucus of the human stomach, NikR plays a more complex regulatory role. To gain insight into how Ni regulates NikR function, we are determining a series of NikR crystal structures and pursuing computational studies to further investigate the significance of our structural findings. Insight gained from these studies will be valuable for our general understanding of intracellular metalloregulation.
