David W. Christianson
Roy and Diana Vagelos Professor in Chemistry and Chemical Biology
Office: 2001 IAST
Lab: 2070 IAST
Phone: (215) 898-5714
Research Group Website: http://www.sas.upenn.edu/~chris/
Research StatementWe 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, human arginase II, and Plasmodium falciparum arginase. 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. 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 (Figure 1), 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], and P. falciparum arginase [Dowling et al. (2010) Biochemistry 49 5600]
Figure 1: Human
arginase I-ABH complex. (a) Omit electron density map of ABH bound in the
enzyme active site at 1.29 Ĺ 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 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 such as atherosclerosis [Santhanam et al. (2007) Circulation Res. 101, 692; Ryoo et al. (2008) Circulation Res. 102, 923]. The biopharmaceutical company Arginetix was founded in 2008 based on our arginase inhibitor technology.
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 [Dowling et al. (2008) Biochemistry 47, 13554; Dowling et al. (2010) Biochemistry 49, 5048] and polyamine deacetylase [Lombardi et al. (2011) Biochemistry 50, 1808.]
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] (Figure 2). 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. 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)[Cole et al. (2011) Biochemistry 50, 258.]
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 60,000 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; Christianson (2008) Curr. Opin. Chem. Biol. 12, 141]. We have determined the three-dimensional crystal structures of terpenoid cyclases from various bacterial, fungal, and plant sources, such as epi-isozizaene synthase from S. colicolor [Aaron et al. (2010) Biochemistry 49, 1787], 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], trichodiene synthase from F. sporotrichioides [Rynkiewicz et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13543], δ-cadinene synthase from cotton [Gennadios et al. (2009) Biochemistry 48, 6175] and taxadiene synthase from the Pacific yew (which catalyzes the first committed step in the biosynthesis of Taxol, a potent cancer chemotherapeutic compound), [Köksal et al. (2011) Nature 469, 116]. To illustrate; structures of bornyl diphosphate synthase and taxadiene synthase are shown in Figures 3 and 4, respectively. 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].
Figure 4: Structural relationships among terpenoid cyclases. The class I terpenoid cyclase fold of pentalenene synthase (blue) contains metal-binding motifs DDXXD and (N,D)DXX(S,T)XXXE (red and orange, respectively); in 5-epi-aristolochene synthase, this domain is linked to a smaller, vestigial domain (green). A related b-domain is found in the class II terpenoid cyclase fold of squalene-hopene cyclase, where it contains the general acid motif DXDD (brown) and a second domain (yellow) inserted between the first and second helices; a hydrophobic plateau flanking helix 8 (gray stripes) enables membrane insertion. Taxadiene synthase contains both class I and class II terpenoid cyclase folds, but only the class I domain is catalytically active. The role of N-termini (purple) in class I plant cyclases is to "cap" the active site, as shown for 5-epi-aristolochene synthase.
Education and Academic History
- A.B. Harvard College (1983)
- A.M. Harvard University (1985)
- Ph.D. Harvard University (1987)
- Searle Scholar Award (1989-1992)
- Young Investigator Award, Office of Naval Research (1989-1992)
- Alfred P. Sloan Foundation Research Fellow (1992-1994)
- Camille and Henry Dreyfus Teacher-Scholar Award (1993-1994)
- Pfizer Award in Enzyme Chemistry (1999)
- Fellow in Natural Sciences (Chemistry), Sidney Sussex College, University of Cambridge, 2006.
- Underwood Fellowship, Department of Biochemistry, University of Cambridge, 2006 - 2007.
- Senior Fellow, American Asthma Foundation, 2006.
- Fellow of the John Simon Guggenheim Memorial Foundation, 2006 - 2007.
Selected PublicationsChambers, J.M.; Hill, P.A.; Aaron, J.A.; Han, Z.; Christianson, D.W.; Kuzma, N.N.; Dmochowski, I.J. "Cryptophane Xenon-129 Nuclear Magnetic Resonance Biosensors Targeting Human Carbonic Anhydrase". J. Am. Chem. Soc. 2009, 131, 563-569.
Shishova, E.Y.; Di Costanzo, L.; Emig, F.A; Ash, D.E.; Christianson, D.W. "Probing the Specificity Determinants of Amino Acid Recognition by Arginase". Biochemistry 2009, 48, 121-131.
Di Costanzo, L.; Drury, J.E.; Christianson, D.W.; Penning, T.M. "Structure and Catalytic Mechanism of Human Steroid 5β-Reductase (AKR1D1). Mol. Cell Endocrinol. 2009, 301, 191-198.
Di Costanzo, L.; Penning, T.M.; Christianson, D.W. "Aldo-Keto Reductases in which the Conserved Catalytic Histidine is Substituted". Chem.-Biol. Interactions 2009, 178, 127-133.
Gennadios, H.A.; Gonzalez, V.; Di Costanzo, L.; Li, A.; Yu, F.; Miller, D.J.; Allemann, R.K.; Christianson, D.W. "Crystal Structure of (+)-δ-Cadinene Synthase from Gossypium arboreum and Evolutionary Divergence of Metal Binding Motifs for Catalysis". Biochemistry 2009, 48, 6175-6183.
Drury, J.E.; Di Costanzo, L.; Penning, T.M.; Christianson, D.W. "Inhibition of Human Steroid 5β-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex". J. Biol. Chem. 2009, 284, 19786-19790 (Accelerated Publication).
Kim, J.H.; Bugaj, L.; Oh, Y.J.; Bivalacqua, T.; Ryoo, S.; Soucy, K.G.; Santhanam, L.; Webb, A.; Camara, A.; Sikka, G.; Nyhan, D.; Shoukas, A.; Ilies, M.; Christianson, D.W.; Champion, H.C.; Berkowitz, D.E. "Arginase Inhibition Restores NOS Coupling and Reverses Endothelial Dysfunction and Vascular Stiffness in Old Rats". J. Appl. Physiol. 2009, 107, 1249-1257.
Aaron, J.A.; Lin, X.; Cane, D.E.; Christianson, D.W. "Structure of Epi-Isozizaene Synthase from Streptomyces coelicolor A3(2), a Platform for New Terpenoid Cyclization Templates". Biochemistry 2010, 49, 1787-1797.
Di Costanzo, L.; Ilies, M.; Thorn, K.J.; Christianson, D.W. "Inhibition of Human Arginase I by Substrate and Product Analogues". Arch. Biochem. Biophys. 2010, 496, 101-108.
Ilies, M.; Di Costanzo, L.; North, M.L.; Scott, J.A.; Christianson, D.W. "2-Amino-imidazole Amino Acids as Inhibitors of the Binuclear Manganese Metalloenzyme Human Arginase I". J. Med. Chem. 2010, 53, 4266-4276.
Zakharian, T.Y.; Christianson, D.W. "Design and Synthesis of C60-Benzenesulfonamide Conjugates". Tetrahedron Lett. 2010, 51, 3645-3648.
Herbert, De'B.R.; Orekov, T.; Rolonson, A.; Ilies, M.; Perkins, C.; O'Brien, W.; Cederbaum, S.; Christianson, D.W.; Zimmermann, N.; Rothenberg, M.E.; Finkelman, F.D. "Arginase I Suppresses IL-12/IL-23p40 Driven Intestinal Inflammation during Acute Schistosomiasis". J. Immunol. 2010, 184, 6438-6446.
Dowling, D.D.; Gattis, S.G.; Fierke, C.A.; Christianson, D.W. "Structures of Metal-Substituted Human Histone Deacetylase 8 Provide Mechanistic Inferences on Biological Function". Biochemistry 2010, 49, 5048-5056.
Dowling, D.D.; Ilies, M.; Olszewski, K.L.; Portugal, S.; Mota, M.M.; Llinás, M.; Christianson, D.W. "Crystal Structure of Arginase from Plasmodium falciparum and Implications for L-Arginine Depletion in Malarial Infection. Biochemistry 2010, 49, 5600-5608.
Smith, A.B., III.; Xiong, H.; Charnley, A.K.; Brenner, M.; Mesaros, E.F.; Kenesky, C.S.; Di Costanzo, L.; Christianson, D.W.; Hirschmann, R. "Design, Synthesis and Structural Analysis of D,L-Mixed Polypyrrolinones 2: Macrocyclic Hexapyrrolinones". Org. Lett. 2010, 12, 2994-2997.
Aaron, J.A.; Christianson, D.W. "Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases". Pure Appl. Chem. 2010, 82, 1585-1597.
Köksal, M.; Zimmer, I.; Schnitzler, J.-P.; Christianson, D.W. "Structure of Isoprene Synthase Illuminates the Chemical Mechanism of Teragram Atmospheric Carbon Emission". J. Mol. Biol. 2010, 402, 363-373 (cover article).
Köksal, M.; Jin, Y.; Coates, R.M.; Croteau, R.; Christianson, D.W. "Taxadiene Synthase Structure and Evolution of Modular Architecture in Terpene Biosynthesis". Nature, advanced online publication 15 December 2010, doi:10.1038/nature09628. Print publication: Nature 2011, 469, 116-120.
Cole, K.E.; Gattis, S.G.; Angell, H.D.; Fierke, C.A.; Christianson, D.W. "Structure of the Metal-Dependent Deacetylase LpxC from Yersinia enterocolitica Complexed with the Potent Inhibitor CHIR-090". Biochemistry 2011, 50, 258-265.
Lombardi, P.M.; Angell, H.D.; Whittington, D.A.; Flynn, E.F.; Rajashankar, K.R.; Christianson, D.W. "Structure of Prokaryotic Polyamine Deacetylase Reveals Evolutionary Functional Relationships with Eukaryotic Histone Deacetylases". Biochemistry 2011, 50, 1808-1817
Köksal, M.; Hu, H.; Coates, R.M.; Peters, R.J.; Christianson, D.W. "Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase". Nature Chem. Biol., in press.