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Dr. David W. Christianson - Roy and Diana Vagelos Professor in Chemistry and Chemical Biology |
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| X-ray crystallography is a powerful technique of structural
biology which can be used to visualize the three-dimensional structures
of biologically-important macromolecules such as proteins. We use X-ray
crystallography as a tool to probe and engineer the structure and function
of novel proteins. Then, we use chemistry to bridge structural biology
and medicine.
Structural and Mechanistic Studies of Metalloenzymes We are interested in structural aspects of the mechanisms of hydrolytic metalloenzymes such as carbonic anhydrase II and Mn2+2-arginase. In genetic-structural studies of the carbonic anhydrase II active site, we have determined the minimum size and shape of the hydrophobic pocket required for substrate association, and we have determined the importance of hydrogen bond networks with zinc-bound hydroxide for its chemical reactivity. The structures of enzyme-inhibitor complexes yield additional insight on functionally-important residues. With arginase, we have determined the structure of the native enzyme from murine liver and we have used this structure to explore the chemistry and biology of the binuclear manganese cluster. The three-dimensional structure of arginase is consistent with a metal-activated hydroxide mechanism for arginine hydrolysis, where both manganese ions serve to activate the catalytic nucleophile. The design, synthesis, and evaluation of boronic acid analogues of arginine yield the tightest-binding inhibitors of arginase known to date. Crystal structure determinations of arginase complexed with these boronic acid inhibitors reveal the binding of the tetrahedral boronate anion, which mimics the tetrahedral intermediate (and its flanking transition states) in the proposed arginase mechanism:
Legend Arginase-ABH complex. (a) Omit
electron density map of ABH in the arginase active site averaged over the
two monomers in the asymmetric unit and averaged over the two twin domains
A and B as described in the text. The map is contoured at 7.7s and
selected active site residues are indicated. Atoms are color-coded
as follows: C = yellow, O = red, N = blue, B = pale green; water
molecules appear as red spheres. Figure generated with BOBSCRIPT
and Raster3D34,35. (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. Cox
et al., Nature Strct. Biol.1999,6 1043-1047.
From the basis of the structural biology and chemistry, we are exploring the physiology of arginase in regulating arginine bioavailability for nitric oxide (NO) biosynthesis - can arginase inhibition enhance NO biosynthesis and NO-dependent processes? We find that the arginase inhibitor (S)-2-amino-6-boronohexanoic acid enhances relaxation of gastrointestinal smooth muscle and penile smooth muscle. Importantly, smooth muscle relaxation in the corpus cavernosum of the mammalian penis is necessary for erection, so human penile arginase is a potential target for the development of new therapies in the treatment of erectile dysfunction http://cnn.com/HEALTH/men/9910/27/erection.drug/index.html Research with arginase is continuing with the crystal structure determinations of important site-specific variants as well as enzyme-inhibitor complexes. We are using detailed crystal structure information to develop even tighter-binding inhibitors, and these inhibitors may similarly be useful in medicine. Also underway or planned are the crystal structure determinations of human arginase II (the smooth muscle isozyme) as well as agmatinase, a related manganese metalloenzyme. Structural Basis of Terpenoid Biosynthesis Terpenes comprise a family of natural products numbering into the thousands, and members of this family 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. In particular, how do such terpenoid cyclases guide the synthesis of dramatically different cyclization products starting with a common, acyclic precursor? How do these enzymes control product stereochemistry so precisely in the cyclization cascade, and what do the structures of these enzymes tell us about the evolution of biosynthetic pathways? As the first step toward answering these questions, we are determining the three-dimensional crystal structures of several plant, animal, and bacterial cyclases. These include monoterpene cyclases (e.g., (+)- and (Ñ)-bornyl diphosphate synthases), sesquiterpene cyclases (e.g., pentalenene synthase, aristolochene synthase, trichodiene synthase), diterpene cyclases (e.g., taxadiene synthase in the Taxol biosynthetic pathway), and triterpene cyclases (e.g., lanosterol synthase in the cholesterol biosynthetic pathway). Additionally, we are studying catalytic antibodies that cyclize polyene substrates. These enzyme structures will illuminate the molecular basis for specificity and stereochemistry in carbocation-mediated cyclization reactions.
We are also interest in biosynthetic enzymes that additionally process terpenes and other olefins of natural or human origin. Epoxides are chemically reactive moieties that are formed in vivo by oxidation of carbon-carbon double bonds; many epoxides are mutagenic and carcinogenic since they readily alkylate the genetic material. Our recent crystal structure determination of mammalian epoxide hydrolase reveals a dimeric domain-swapped architecture. This enzyme activates and hydrolyzes organic epoxides to form vicinal 1,2-diols, which are generally less mutagenic and more readily excreted due to increased solubility. Unlike its bacterial and fungal counterparts, mammalian epoxide hydrolase contains two domains and each contains an active site ? but only one, the C-terminal domain, participates in epoxide hydrolysis. We are now probing the structural and functional importance of the N-terminal domain in order to discover the evolutionary imperative that linked these two enzyme domains. The figure below shows the domain-swapped epoxide hydrolase dimer.
Bound inhibitors indicate the C-terminal domain implicated in epoxide hydrolysis
(blue); the N-terminal domain of unknown catalytic function (green) is
connected by a 16-residue, proline-rich linker (red).
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