Uniformed Services University of the Health Sciences
Department of Biochemistry and Molecular Biology
4301 Jones Bridge Road, C1094
Bethesda, Maryland 20814-4799
Office Rm. B-4062:
Office Phone: (301) 295-9419
Lab Rm. B-4055
Fax: (301) 295-3512
The research in my lab aims to understand biologically important processes by revealing the complex relationships between the structure, function, and dynamics of proteins through a structural enzymology approach. We take advantage of a combination of x-ray crystallography, biochemical, kinetic, and thermodynamic techniques to describe the functional pathway of biologically relevant molecules. Specifically, my lab focuses on proteins in three processes essential to human health:
Homocysteine (Hcy) is an essential metabolite critical for methionine, S-adenosylmethionine (SAM) and cysteine biosynthesis. Elevated levels of Hcy (hyperhomocysteinemia) pose an increased risk for a number of diseases including cardiovascular diseases, making the regulation of cellular Hcy vital to human health. In mammals, four enzymes control Hcy homeostasis: methionine synthase (MS), cystathionine β-synthase (CBS), and betaine-homocysteine S-methyltransferase (BHMT and BHMT2). My lab studies the structure and function of all four of these enzymes.
Fig. 1 Homocysteine metabolic pathway.
In the one carbon (methionine) cycle Hcy is a product of the transmethylation reactions. Hcy either enters the transsulfuration pathway (via CBS) to eventually form glutathione, or is remethylated to methionine by MS or BHMT in the one carbon cycle. MS,couples Hcy metabolism to the folate cycle.
Methionine synthase (MS). Methionine synthase is a multi-domain vitamin B12-dependent enzyme whose function relies on the delivery of vitamin B12 through a complex B12 maturation/delivery pathway. In mammals, MS is the only Hcy clearing enzyme expressed in all tissue types, and the only such enzyme found in vascular tissue. MS polymorphisms are linked to numerous human diseases and cause megaloblastic anemia. However, the function and regulation of mammalian MS are poorly understood. Moreover, it is unclear how MS polymorphisms disrupt enzyme function. The goal of this work is to reveal the mechanism of human MS catalysis and regulation, providing insight into a variety of MS and Hcy-related human diseases. This work will characterize the relationship between MS function and B12 metabolism, and provide a framework for understanding how MS contributes to Hcy homeostasis. Our studies will reveal which B12 maturation/delivery proteins might make suitable targets for new therapeutics against the cardiovascular disease risk factor hyperhomocysteneimia.
Betaine-homocysteine S-methyltransferases (BHMT and BHMT2). BHMT and BHMT2 are zinc dependent mammalian enzymes that catalyze the same reaction as MS, but with remarkably different chemistry and protein scaffolds. In addition to maintaining Hcy levels, these proteins are postulated to contribute to lipid and phospholipid metabolism. Through a combination of structural enzymology, chemical biology, and in vivo approaches, we are working to uncover the structure, mechanism, and precise physiological roles of BHMT and BHMT2 in vivo.
Fig. 2 Structure of CBS from Drosophila shown in a dimer pair.
Cysteine (Cys) biosynthesis via the transsulfuration pathway is an integral piece of the Hcy degradation pathway. The production of Cys is not only essential for Hcy catabolism, but also for the synthesis of glutathionine (GSH), an abundant small thiol molecule critical for the protection of cells from oxidative damage and maintaining redox homeostasis. The transsulfuration of Hcy to Cys is catalyzed by the successive action of two enzymes: cystathioninine b-synthase (CBS) and cystathionine g-lyase (CSE). Mutations in CBS are the single most common cause of hereditary hyperhomocysteinemia with > 100 mutations having been observed in patients. In addition to converting Hcy to Cys, these two enzymes are responsible for the biosynthesis of the newly discovered signaling molecule H2S, which is important for the cardiovascular and nervous systems. Recently, a third enzyme, 3-mercaptopyruvate sulfurtransferase (3MST), was identified as an additional contributor to H2S production. H2S serves a number of roles such as acting as a neuromodulator, regulator of hormone release, vasorelaxative, preserver of mitochondrial function, endogenous modulator of inflammation, and increasing levels of GSH by activating cystine transport. Unlike other gasotransmitters (eg. CO and NO) the vascular effects, mechanism of action, synthesis, and regulation of H2S are not well studied. The aim of this project is to understand the fundamentals of H2S biosynthesis and regulation through the investigation of the enzymes of the transsulfuration pathway involved in its production.
Mitochondrial (mt) tRNA genes are hot spots for mutations that lead to adverse health effects with a wide range of pathology, from isolated organ-specific diseases such as myopathy or hearing loss, to multisystem disorders with encephalopathy, gastrointestinal dysmotility, and life-threatening cardiomyopathy. All tRNAs are regularly interspersed throughout the entire compact mitochondrial genome and require the combined action of two mitichondrial endonucleases, a 5' and 3' end one in order to be excised. Mitochondrial tRNA processing is important not only for the maturation and release of tRNAs but also of for the maturation and release of of rRNAs and mRNAs. Mutations in tRNA genes may impact multiple steps during a tRNA's life cycle, including 5' end maturation by mitochondrial ribonuclease P (mtRNaseP). RNase P is responsible for catalyzing tRNA 5' end maturation across all three domains of life. Our goal is to investigate this maturation process since we believe it provides a mechanistic link to mitochondrial dysfunction and aging.
Fig. 3 Structure of mt PNase P from Arabidopsis thaliana with precursor tRNA substrate modeled in.
Until recently, all known RNase P enzymes included a catalytic RNA component. The discovery of a protein-only RNase P (mt RNase P or MRPP3) from human mitochondria and A. thaliana chloroplast and mitochondria shifted this paradigm, with these enzymes representing a new class of metallonucleases. MRPP3s are conserved among higher eukaryotes and evolved recently as yeast mitochondrial genomes encode RNase P with a catalytic RNA. We recently solved the structure of mt RNase P from A. thaliana revealing an N-terminal PPR domain and a C-terminal metallonuclease PIN-like domain. In higher eukaryotes two additional proteins were identified as important for in vivo MRPP3 activity (MRPP1 and MRPP2). We hypothesize that the three MRPP proteins are essential for mitochondrial viability and provide a link between RNA processing and mitochondrial diseases and aging. We are employing a highly collaborative in vivo and in vitro approach to study the structure, mechanism and role of MRPPs in biological processes.