Abstract(+)-δ-Cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 Å resolution and the structure of its complex with three putative Mg 2+ ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 Å resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich motif D 307 DTYD 311 on helix D that interacts with Mg 2+A and Mg 2+ C . However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg 2+ B , which is usually found as the signature sequence (N, D)D(L, I, V)X(S, T)XXXE (boldface indicates Mg 2+ B ligands). Instead, DCS contains a second aspartate-rich motif, D 451 DVAE 455 , that interacts with Mg 2+ B . In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg 2+ B -binding motif. Analyses of DCS mutants with alanine substitutions in the D 307 DTYD 311 and D 451 DVAE 455 segments reveal the contributions of these segments to catalysis.Antimicrobial natural terpenoid products known as phytoalexins are generated by tree cotton (Gossypium arboreum, indigenous to India and Pakistan) in response to threats from bacterial or fungal pathogens (1-2). These products are classified as secondary metabolites and are † This work was supported by National Institutes of Health grant GM56838 (D.W.C.), BBSRC grants 6/B17177 (R.K.A.) and BB/ G003572/1 (R.K.A), EPSRC grant EP/D06958/1 (R.K.A), Royal Society grant 2007R2 (R.K.A.), and Cardiff University. ‡ The atomic coordinates of unliganded (+)-δ-cadinene synthase and its complex with 2-fluorofarnesyl diphosphate have been deposited in the Protein Data Bank (www.rcsb.org) with accession codes 3G4D and 3G4F, respectively. Δ These authors made equal contributions to this study. * To whom correspondence should be addressed: DWC: Tel: 215-898-5714. Fax: 215-573-2201. Email: chris@sas.upenn NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript therefore non-essential for plant viability; however, they mediate important interactions between plants and their environments (3). The 64 kDa sesquiterpenoid cyclase (+)-δ-cadinene synthase (DCS, isozyme XC1; SWISS-PROT accession code Q39761) catalyzes the formation of (+)-δ-cadinene from farnesyl diphosphat...
The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 Å resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor, TU-514, and the 2.0 Å resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence is also presented for a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.The outer leaflet of the outer membrane of a Gram-negative bacterium is composed of lipopolysaccharide (LPS), which serves as a permeability barrier that protects the bacterium against erythromycin and other antibiotics (1-6). 1 Each LPS molecule contains O-antigen † This work was supported by National Institutes of Health grants GM49758 (D. W. C.) and GM40602 (C. A. F.) ‡ The atomic coordinates for LpxC complexed with TU-514 and imidazole have been deposited in the Protein Data Bank, www.rcsb.org, with accession codes 2GO4 and 2GO3, respectively. * To whom correspondence should be addressed. Telephone: (215) 898-5714. Fax: (215) 573-2201. Email: chris@sas.upenn.edu. § University of Pennsylvania ⊥ Current address: Amgen, Inc., One Kendall Square, Bldg. 1000, Cambridge, MA 02139 ‖ University of Alberta; current address: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA 02138 # University of Michigan 1 Abbreviations: LPS, lipopolysaccharide; TU-514, 1,5-anhydro-2-C-(carboxymethyl-N-hydroxamide)-2-deoxy-3-O-myristoyl-Dglucitol; UDP, uridine 5′-diphosphate; NCS, noncrystallographic symmetry; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic ...
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