David W. Christianson

The atomic-resolution crystal structures of human carbonic anhydrases I and II complexed with “two-prong” inhibitors are reported. Each inhibitor contains a benzenesulfonamide prong and a cupric iminodiacetate (IDA-Cu²⁺) prong separated by linkers of different lengths and compositions. The ionized NH^- group of each benzenesulfonamide coordinates to the active site Zn²⁺ ion; the IDA-Cu²⁺ prong of the tightest-binding inhibitor, BR30, binds to H64 of CAII and H200 of CAI. This work provides the first evidence verifying the structural basis of nanomolar affinity measured for two-prong inhibitors targeting the carbonic anhydrases.

X‐ray crystal structures of human soluble epoxide hydrolase (sEH) complexed with four different dialkylurea inhibitors bearing pendant carboxylate “tails” of varying length have been determined at 2.3–3.0 Å resolution. Similarities among inhibitor binding modes reinforce the proposed roles of Y381 and/or Y465 as general acids that protonate the epoxide ring of the substrate in concert with nucleophilic attack of D333 at the electrophilic epoxide carbon. Additionally, the binding of these inhibitors allows us to model the binding mode of the endogenous substrate 14,15‐epoxyeicosatrienoic acid. Contrasts among inhibitor binding modes include opposite orientations of inhibitor binding in the active‐site hydrophobic tunnel. Alternative binding orientations observed for this series of inhibitors to human sEH, as well as the binding of certain dialkylurea inhibitors to human sEH and murine sEH, complicate the structure‐based design of human sEH inhibitors with potential pharmaceutical applications in the treatment of hypertension. Thus, with regard to the optimization of inhibitor designs targeting human sEH, it is critical that human sEH and not murine sEH be utilized for inhibitor screening, and it is critical that structures of human sEH‐inhibitor complexes be determined to verify inhibitor binding orientations that correlate with measured affinities.

Human arginase I is a potential target for therapeutic intervention in diseases linked to compromisedl-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH,K_d= 5 nM) andS-(2-boronoethyl)-l-cysteine (BEC,K_d= 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme–inhibitor complexes at 1.29- and 1.94-Å resolution determined from crystals twinned by hemihedry. The ultrahigh-resolution structure of the human arginase I–ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate–manganese coordination interactions and fully saturated hydrogen bond networks with inhibitor α-amino and α-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state forl-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group ofl-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.

The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP_i) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP_i bind three Mg²⁺ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg²⁺₃-PP_i, in which R304 donates hydrogen bonds to PP_i and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP_i in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP_i and (S)-azabisabolene. Thus, although the R304−D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg²⁺₃-PP_i binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in k_cat/K_M, it is clear that a properly formed R304−D101 hydrogen bond is required in the enzyme−substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP_i leaving group.

The outer leaflet of the outer membrane of the Gram-negative bacterium serves as a permeability barrier and is composed of lipopolysaccharide, also known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the biosynthesis of which is essential for cell viability. The first committed step in lipid A biosynthesis is catalyzed by UDP-(3- O -( R -3-hydroxymyristoyl))- N -acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase. Here we report the crystal structure of LpxC from Aquifex aeolicus , which reveals a new α+β fold reflecting primordial gene duplication and fusion, as well as a new zinc-binding motif. The catalytic zinc ion resides at the base of an active-site cleft and adjacent to a hydrophobic tunnel occupied by a fatty acid. This tunnel accounts for the specificity of LpxC toward substrates and inhibitors bearing appropriately positioned 3- O -fatty acid substituents. Notably, simple inhibitors designed to target interactions in the hydrophobic tunnel bind with micromolar affinity, thereby representing a step toward the structure-based design of a potent, broad-spectrum antibacterial drug.

The x-ray crystal structure of dimeric (+)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis , is reported at 2.0-Å resolution. Each monomer contains two α-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis. Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide “snapshots” of the terpene cyclization cascade.

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC 4.2.1.1 ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning α-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-Å resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix–helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby α-helical “130's segment.” The structure of the CA XII–acetazolamide complex is also reported at 1.50-Å resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.

The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3 ) has been determined at 2.8-Å resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the α/β hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.