Dr. David Christianson
Roy and Diana Vagelos Professor in Chemistry and Chemical Biology
Roy and Diana Vagelos Laboratories
Department of Chemistry
University of Pennsylvania
231 South 34th Street
Philadelphia, PA 19104
(215) 898 5714 (office)
(215) 573 2201 (fax)
X-ray crystallography is a powerful technique of structural biology which can be used to visualize the structures of biologically important macromolecules such as proteins. My research interests focus mainly on metal-requiring enzymes, and we use X-ray crystallography to determine their crystal structures. Enzyme structures provide inferences on catalytic mechanism and also guide the design of enzyme inhibitors. Additionally, we use chemical approaches to probe and regulate the activity of metal-requiring enzymes in vitro and in vivo. Our studies are highly interdisciplinary and span the fields of chemistry, biology, and medicine. For example, we have used techniques of structural and chemical biology in the discovery of a new drug target for the treatment of human sexual arousal disorders, and one of the enzyme inhibitors emanating from our structure-based design program is being developed as a drug for the treatment of cardiovascular disorders. In other work, we have illuminated the structural basis of biosynthetic diversity in the family of terpenoid synthases, metal-requiring enzymes responsible for the generation of thousands of natural products in all forms of life.
Structural and Mechanistic Studies of Hydrolytic Metalloenzymes
We are interested in structural aspects of the mechanisms of hydrolytic metalloenzymes such as the arginases. To date, we have determined the crystal structures of rat arginase I, human arginase I, and human arginase II. Structural and enzymological data suggest a mechanism for arginine hydrolysis in which both manganese ions activate a bridging hydroxide ion for nucleophilic attack at the guanidinium group of arginine in the first step of catalysis. Based on our structural and mechanistic analyses, we designed and synthesized boronic acid analogues of arginine such as 2-amino-6-boronohexanoic acid (ABH, Kd = 5 nM) [Baggio et al. (1997) J. Am. Chem. Soc. 1997, 119, 8107]. The boronic acid moiety of ABH similarly undergoes nucleophilic attack by the metal-bridging hydroxide ion to yield a metal-bound boronate anion that mimics the tetrahedral intermediate and its flanking transition states in catalysis, as shown in X-ray crystallographic studies of rat arginase I [Cox et al. (1999) Nature Struct. Biol. 6, 1043] and human arginase I [Di Costanzo et al. (2005) Proc. Natl. Acad. Sci. USA, 102, 13058].
Figure 1: Human arginase I-ABH complex. (a) Omit electron density map of ABH bound in the enzyme active site at 1.29 Ã… resolution. Water molecules appears as red spheres and Mn(II) ions appears as larger pink spheres. (b) Summary of arginase-ABH interactions; manganese coordination interactions are designated by green dashed lines, and hydrogen bonds are indicated by black dashed lines. (c) Stabilization of the tetrahedral intermediate (and flanking transition states) in the arginase mechanism based on the binding mode of ABH.
We have also used ABH as a chemical tool for probing the role of arginase in regulating arginine bioavailability for nitric oxide (NO) biosynthesis in tissues and in live animals. We discovered that arginase inhibition by ABH enhances smooth muscle relaxation in ex vivo organ bath studies. Since smooth muscle relaxation in the corpus cavernosum of the penis is necessary for erection, we concluded that human penile arginase is a potential target for the development of new therapies in the treatment of erectile dysfunction [Cox et al. (1999) Nature Struct. Biol. 6, 1043]. Our subsequent in vivo studies demonstrated that arginase inhibition by ABH enhances erectile function and vasocongestion in the male and female genitalia, so we concluded that both male erectile dysfunction and female sexual arousal disorder are potentially treatable by ABH [Cama et al. (2003) Biochemistry 2003, 42, 8445; Christianson (2005) Acc. Chem. Res. 38, 191]. More recent studies show that ABH may also be useful in the treatment of certain cardiovascular disorders [Santhanam et al. (2007) Circulation Res. 2007, 101, 692].
Research with arginase is continuing with the crystal structure determinations of important site-specific variants as well as enzyme-inhibitor complexes. For example, we have recently shown that the C=S moiety of thiosemicarbazide is capable of bridging the binuclear manganese cluster in the arginase active site [Di Costanzo et al. (2007) J. Am. Chem. Soc. 129, 6388]. We are also preparing for the neutron crystal structure determination of human arginase I, which should allow us to pinpoint the locations of specific protons important for binding and catalysis [Di Costanzo et al. (2007) Arch. Biochem. Biophys. 465, 82]. Additionally, we are studying the crystal structures of bacterial arginases as well as enzymes that adopt the arginase fold, such as histone deacetylase and polyamine deacetylase.
In other metalloenzyme work, we have determined the crystal structure of A. aeolicus LpxC, a zinc-requiring enzyme that catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria [Whittington et al. (2003) Proc. Natl. Acad. Sci. USA 100, 8146]. Subsequent structural studies have allowed us to pinpoint regions of the active site that interact with the fatty acid and diphosphate moieties of the substrate [Gennadios et al. (2006) Biochemistry 45, 7940; 15216], and these studies have guided the first steps in the structure-based design of new LpxC inhibitors that may ultimately be useful in the treatment of Gram-negative bacterial infections [Shin et al. (2007) Bioorg. Med. Chem. 2007, 15, 2617]. To date, we have broadened these structural studies to include LpxC enzymes from Gram-negative pathogens Y. pestis (bubonic plague) and F. tularensis (tularemia).
Figure 2: Structure and biological function of LpxC. This zinc enzyme catalyzes the first committed step of lipid A biosynthesis; lipid A is the hydrophobic anchor of lipopolysaccharide, which comprises the outer leaflet of the outer membrane of Gram-negative bacteria. The crystal structure of LpxC reveals a hydrophobic tunnel in the active site that accommodates the fatty acid moiety of the substrate, and this binding interaction is required for the active site to adopt a catalytically-active conformation.
Structural Basis of Terpenoid Biosynthesis
The family of terpenoid natural products currently numbers more than 55,000 members found in all forms of life. Terpenoids, are involved in diverse biological functions such as the mediation of plant-parasite interactions or the modulation of membrane fluidity. Since times of antiquity, terpenoid natural products have also been essential components of the pharmacopeia as analgesics, antibiotics, and anti-cancer compounds (e.g., Taxol). We are interested in the enzymes that catalyze the biosynthesis of different cyclic terpenoids [Christianson (2006) Chem. Rev. 106, 3412]. We have determined the three-dimensional crystal structures of terpenoid cyclases from various bacterial, fungal, and plant sources, such as bornyl diphosphate synthase from culinary sage [Whittington et al. (2002) Proc. Natl. Acad. Sci. USA 99, 15375], aristolochene synthase from A. terreus [Shishova et al. (2007) Biochemistry 46, 1941], and trichodiene synthase from F. sporotrichioides [Rynkiewicz et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13543]; X-ray crystal structure determinations are currently underway for δ-cadinene synthase from cotton and taxadiene synthase from the Pacific yew (which catalyzes the first committed step in the biosynthesis of Taxol, a potent cancer chemotherapeutic compound). These structures guide the study of site-specific mutants and alternative substrates as we explore the structural basis of diversity in terpenoid biosynthesis [e.g., see: Vedula et al. (2005) Biochemistry 44, 12719; Vedula et al. (2007) Arch. Biochem. Biophys. 466, 260; Vedula et al. (2008) Arch. Biochem. Biophys. 469, 184; Christianson (2007) Science 316, 60].
Figure 3: Reaction catalyzed by bornyl diphosphate synthase. Aza analogues of carbocation intermediates are shown in boxes; crystal structures of their complexes with the synthase reveal structural inferences on catalysis. The enzyme undergoes significant conformational changes upon the binding of 3 Mg2+ ions and pyrophosphate (or a substrate diphosphate group). These conformational changes sequester the active site from bulk solvent and trigger substrate ionization to initiate catalysis [Whittington et al. (2002) Proc. Natl. Acad. Sci. USA 99, 15375].
Our most recently determined structure of a terpenoid cyclase is that of aristolochene synthase from A. terreus [Shishova et al. (2007) Biochemistry 46, 1941]. Subsequent studies of substrate binding suggest a specific sequence for the binding of the 3 catalytically obligatory Mg2+ ions. Additionally, as the quicktime movie below illustrates, the binding of the second metal ion triggers significant active site conformational changes from the "open" state to the "closed" state, and these conformational changes are completed upon the binding of the third metal ion:
We are also interest in biosynthetic enzymes that process cyclic terpenoids, including steroids, in biosynthetic reactions further downstream. For example, we have determined X-ray crystal structures of human liver D4-3-Ketosteroid 5b-Reductase (AKR1D1) and we are currently studying its complexes with cofactor NADP+ and steroid substrates such as testosterone. These studies promise to illuminate structural aspects of enzyme mechanism relevant to understanding the molecular basis of hereditary defects in human bile acid biosynthesis.
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