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1.  Crystal Structures of Penicillin-Binding Protein 3 from Pseudomonas aeruginosa: Comparison of Native and Antibiotic-Bound Forms 
Journal of Molecular Biology  2011;405(1-3):173-184.
We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.
PMCID: PMC3025346  PMID: 20974151
PBP, penicillin-binding protein; HMM, high molecular mass; LMM, low molecular mass; PDB, Protein Data Bank; ESRF, European Synchrotron Radiation Facility; anti-bacterial; Pseudomonas aeruginosa; carbenicillin; ceftazidime; enzyme structure
2.  Structure and Reactivity of Bacillus subtilis MenD Catalyzing the First Committed Step in Menaquinone Biosynthesis 
Journal of Molecular Biology  2010;401(2):253-264.
The first committed step in the classical biosynthetic route to menaquinone (vitamin K2) is a Stetter-like conjugate addition of α-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 Å) crystal structure of Bacillus subtilis MenD with cofactor and Mn2+ has been determined. Based on structure–sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of α-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure–reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.
PMCID: PMC2914249  PMID: 20600129
CoA, coenzyme A; PDB, Protein Data Bank; SAD, single-wavelength anomalous diffraction; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate; SeMet, selenomethionine; ThDP, thiamine diphosphate; PEG, polyethylene glycol; crystal structure; enzyme mechanism; menaquinone biosynthesis; thiamine diphosphate cofactor

Results 1-2 (2)