The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 Å resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with 3 Mg2+ ions, inorganic pyrophosphate (PPi), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 Å resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation-π interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 Å resolution crystal structure of F198A EIZS complexed with 3 Mg2+ ions, PPi, and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS complexed with 4 Hg2+ ions has been determined at 1.90 Å resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster not only serves to bind and orient the flexible isoprenoid substrate in the precatalytic Michaelis complex, but it also triggers the departure of the diphosphate leaving group to generate a carbocation that initiates catalysis. Additional conserved hydrogen bond donors assist the metal cluster in this function. Crystal structure analysis reveals that the constellation of three metal ions required for terpenoid synthase catalysis is generally identical among all class I terpenoid synthases of known structure.

The α-carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of CO 2 in forming HCO 3 -. The active site of an α-CA contains a catalytically essential Zn 2+ coordinated by three histidine residues at the bottom of a 15 Å deep cleft, and the tightest binding CA inhibitors developed to date contain a sulfonamide moiety that coordinates to Zn 2+ as a sulfonamidate anion. 1 Notably, human isozyme II (CAII) is an ideal model system for exploring new inhibitor designs, some of which can be exploited in biosensing applications. 2-4 Here, CAII is utilized for the structure-based design of a xenon ( 129 Xe) biosensor for potential use as a magnetic resonance imaging (MRI) contrast agent.The 129 Xe isotope has a spin-1/2 nucleus, a >200-ppm chemical shift window in water, and a natural isotopic abundance of 26% (commercially available up to 86%), which makes it an appealing biomolecular probe for MRI. Moreover, 129 Xe can be laser polarized to enhance MRI signals ∼10,000-fold. 5 Although current in vivo MRI applications are limited to functional lung imaging through the diffusion of Xe gas, 6 the encapsulation of 129 Xe within a cryptophane cage (K D ≈ 30 μM at 37 °C in phosphate-buffered solution) 7 facilitates its use as a biosensor that can be targeted to specific proteins using an appropriate affinity tag. 8,9 For example, racemic biosensor 1 (Figure 1a) has been designed to bind to the CA isozymes (K D = 60 ± 20 nM against CAII in solution), and yields a distinctive 129 Xe-MRI spectrum when bound to CAII. 10 Here, we report the X-ray crystal structure of the CAII-1-Xe complex at 1.70 Å resolution.For structure determination, CAII was overexpressed in E. coli and purified as described, 11 then incubated with a two-fold excess of 1, concentrated to 10 mg/mL, and crystallized by the hanging drop vapor diffusion method. Crystals were cryoprotected in 15% glycerol and subsequently pressurized under 20 atm Xe for 30 min prior to flash cooling and X-ray data collection. The structure was refined to final R work and R free values of 0.23 and 0.25, respectively.Biosensor 1 coordinates to the active site Zn 2+ ion as the sulfonamidate anion, displacing the zinc-bound hydroxide ion of the native enzyme as previously observed in other complexes of CAII with benzenesulfonamide derivatives. 1,2,12 The crystallographic occupancies of 1 and Zn 2+ are refined at 0.5. It is unusual to observe diminished Zn 2+ occupancy in a CAII-inhibitor complex, but the molecular origins of this effect are not clear.The encapsulation of Xe within the cryptophane cage of 1 is confirmed by inspection of the Bijvoet difference Fourier map calculated from anomalous scattering data (Figures 1b and S1.) X-ray diffraction data was collected at a wavelength λ = 0.9795 Å, which is far from the Xe L I edge of 2.27 Å. 13 Nevertheless, the anomalous scattering component f" is 3.4 e -for Xe, so Email: chris@sas.upenn.edu. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2009 June 4. NIH-PA A...

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