Residues involved in ligand binding are shown as sticks and colored cyan

Residues involved in ligand binding are shown as sticks and colored cyan. crucial for microorganisms, plants and fungi. The recent emergence of multi-drug resistant pathogenic microbes demonstrates a pressing need to develop new antibiotics. The absence of the shikimate pathway in humans presents an attractive target in the development of antimicrobials. Numerous studies have attempted ZM 336372 to target enzymes in the shikimate pathway [2]. Currently, N-phosphomethylglycine is the only commercially available compound that targets one of the enzymes in the pathway; it targets 5-enolpyruvate shikimate-3-phosphate synthase Ephb4 [3], [4], [5]. 3-Dehydroquinate dehydratase (DHQase) is the third enzyme in the shikimate pathway. DHQase catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate (Figure 1). There are two types of DHQase: type I enzymes catalyze a Schiff base mechanism using a catalytic lysine ZM 336372 residue; type II DHQase catalyze the dehydration reaction an enolate intermediate. DHQase from is a type I enzyme. Other organisms that have type I DHQases include (efDHQase). The study also elucidated the structure of DHQase to a resolution of 2.2 ?. This study provides significant biochemical and structural information that will facilitate the future development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the nosocomial pathogen (efDHQase) The gene encoding 3-dehydroquinate dehydratase (efDHQase, 3-dehydroquinate dehydratase from V583 strain) (GI: 29376281) was amplified PCR from genomic DNA isolated from V583 strain using Platinum DNA polymerase (Invitrogen). The PCR mixture (100 L) contained 1 ng of plasmid DNA, 10 L of 10 Pfx amplifi cation buffer, 1 mM MgSO4, dNTPs (0.4 mM each), 40 pmol of each primer (forward primer and reverse primer DNA polymerase. The gene was amplified using a PTC-0200G Thermal Cycler (Bio-Rad Laboratories), with the following parameters: 94C for 2 min followed by 40 cycles of 94C for 1 min, 55C for 1 min and 15 s, and 68C for 3 min, and a final extension of 68C for 10 min. The amplified gene was cloned into a modified pET-15b vector (Novagen) in which the N-terminus contained 10 His residues (kindly provided by Professor John Gerlt, University of Illinois, Urbana, IL) [12]. The protein was expressed in negative mutant strain in which the gene was deleted from the genome. Transformed cells were grown at 37C in LB broth (supplemented with 100 g/mL of ampicillin, ZM 336372 15 g/mL of chloramphenicol and 50 g/mL of kanamycin) to an OD600 of 0.6, and IPTG (0.1 mM) was added to induce protein expression for 16 h. The cells were harvested by centrifugation and resuspended in binding buffer [5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9)] and lysed by sonication. The lysate was clarified by centrifugation, and the His-tagged protein was purified using a column of chelating Sepharose Fast Flow (GE Healthcare Bio-Sciences Corp.) charged with Ni2+ ion. The cell lysate was applied to the column in binding buffer, washed with buffer comprising 154 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl, pH 7.9, and eluted with 100 mM L-histidine, 0.5 M NaCl, and 20 mM Tris-HCl, pH 7.9. The ZM 336372 N-terminal His tag was eliminated with thrombin (GE Healthcare Bio-Sciences Corp.) according to the manufacturer’s instructions, and the proteins were purified to homogeneity on a Q Sepharose High Performance column (GE Healthcare Bio-Sciences Corp.) equilibrated with binding buffer [25 mM Tris-HCl, pH 7.9] and eluted having a linear gradient from 0 to 0.5 M elution buffer [1 M NaCl and 25 mM Tris-HCl, pH 7.9]. Cloning, manifestation and purification of shikimate dehydrogenase.