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Orotidine 5'-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate (UMP) by 17 orders of magnitude. Eight new crystal structures with ligand analogues combined with computational analyses of the enzyme's short-lived intermediates and the intrinsic electronic energies to distort the substrate and other ligands improve our understanding of the still controversially discussed reaction mechanism. In their respective complexes, 6-methyl-UMP displays significant distortion of its methyl substituent bond, 6-amino-UMP shows the competition between the K72 and C6 substituents for a position close to D70, and the methyl and ethyl esters of OMP both induce rotation of the carboxylate group substituent out of the plane of the pyrimidine ring. Molecular dynamics and quantum mechanics/molecular mechanics computations of the enzyme-substrate complex also show the bond between the carboxylate group and the pyrimidine ring to be distorted, with the distortion contributing a 10-15% decrease of the ΔΔG(⧧) value. These results are consistent with ODCase using both substrate distortion and transition-state stabilization, primarily exerted by K72, in its catalysis of the OMP decarboxylation reaction.

The structure of neamine bound to the A site of the bacterial ribosomal RNA was used in the design of novel aminoglycosides. The design took into account stereo and electronic contributions to interactions between RNA and aminoglycosides, as well as a random search of 273 000 compounds from the Cambridge structural database and the National Cancer Institute 3-D database that would fit in the ribosomal aminoglycoside-binding pocket. A total of seven compounds were designed and subsequently synthesized, with the expectation that they would bind to the A-site RNA. Indeed, all synthetic compounds were found to bind to the target RNA comparably to the parent antibiotic neamine, with dissociation constants in the lower micromolar range. The synthetic compounds were evaluated for antibacterial activity against a set of important pathogenic bacteria. These designer antibiotics showed considerably enhanced antibacterial activities against these pathogens, including organisms that hyperexpressed resistance enzymes to aminoglycosides. Furthermore, analyses of four of the synthetic compounds with two important purified resistance enzymes for aminoglycosides indicated that the compounds were very poor substrates; hence the activity of these synthetic antibiotics does not appear to be compromised by the existing resistance mechanisms, as supported by both in vivo and in vitro experiments. The design principles disclosed herein hold the promise of the generation of a large series of designer antibiotics uncompromised by the existing mechanisms of resistance.

Matrix metalloproteinases (MMPs) are important hydrolytic enzymes with profound physiological and pathological functions in living organisms. MMPs are produced in their inactive zymogenic forms, which are subsequently proteolytically activated in an elaborate set of events. The propeptide in the zymogen blocks the active site, with a cysteine side-chain thiolate from this propeptide achieving coordination with the catalytically important zinc ion in the active site. Molecular dynamics simulations, ab initio calculations, and wet chemistry experiments presented herein argue for the critical importance of a protonation event at the coordinated thiolate as a prerequisite for the departure of the propeptide from the active site. Furthermore, a catalytically important glutamate is shown to coordinate transiently to the active-site zinc ion to "mask" the positive potential of the zinc ion and lower the energy barrier for dissociation of the protonated cysteine side chain from the zinc ion. In addition, a subtle conformational change by the propeptide is needed in the course of zymogen activation. These elaborate processes take place in concert in the activation process of MMPs, and the insight into these processes presented herein sheds light on a highly regulated physiological process with profound consequences for eukaryotic organisms. Proper interactions of eukaryotic cells with the extracellular matrix (ECM) are important for the normal functions within a tissue. ECM-cell interactions are regulated by a family of at least 27 zinc-dependent endopeptidases known as matrix met- alloproteinases (MMPs), the functions of which have profound consequences in organ development, tissue remodeling, em- briogenesis, wound healing, and angiogenesis. 1 Because of the critical functions of these enzymes in many physiological processes, their activities are strictly regulated. 2 Each is produced as an inactive zymogen form (pro-MMP), which requires activation for generation of the catalytically competent enzyme. The process of zymogen activation appears to be complicated. 3,4 A second level of control of MMP activity is exerted by inhibition of the active enzymes by a group of four specific proteins known as the tissue inhibitors of metalloproteinases ("TIMPs"). When these elaborate regulatory processes fail, the

Lipopolychacharides (LPSs) cover the surface of Gram-negative bacteria. The LPS assembly provides a penetration barrier to molecules larger than 700−1000 Da, such as many antibiotics, and when LPS molecules are released from the surface of bacteria, they cause toxic shock in the infected patient. The surface of Escherichia coli JM109 was visualized by atomic force microscopy, providing the highest resolution images of any bacterium to date (50-A lateral and 5-A vertical resolutions). These images indicated that LPS molecules are assembled in bundles of 600−3500 molecules. The LPS molecule is comprised of lipid A, then an inner and outer core, and an outermost region of O-antigen units. Analyses indicated that the O-antigen repeat units vary between 1 and 26 for E. coli JM109, and the fraction of the LPS molecules with zero to three repeat units made up approximately 50% of the total LPS content. A matrix of 16 LPS molecules was constructed as a representative region of the surface of E. coli. The molecula...

Hydrogen bonds between a small molecule and an enzyme can potentially contribute significantly to the stability of the complex. Such electrostatic interactions can also lower energy barriers for reactions by solvation of high-energy species. A novel type of inhibitor is described in this report, which was designed to take advantage of a hydrogen bond that it makes to the active-site histidine of chymotrypsin to attenuate its basicity. Substrates of chymotrypsin acylate the active-site serine (of the catalytic triad), and the acyl-enzyme intermediate undergoes deacylation in a second step of the catalytic turnover. The active-site histidine (of the catalytic triad) serves as the general base in both steps of the turnover process. Such attenuation of basicity by hydrogen bonding was expected to impair catalysis by the enzyme. Two molecules of this type were synthesized that are based on the structure of the chymotrypsin substrate Ac-l-Ala-l-Ala-Gly-l-Phe methyl ester. These were methyl (2R,3R)-5-(N-acetyl-l...

International audience; Bacteria resistant to antibiotics are being selected in a relatively short time, and cases of infections resistant to treatment by all known antibiotics are being identified at alarming rates. The primary mechanism for resistance to-lactam antibiotics is the catalytic function of-lactamases. However, imipenem (a-lactam) resists the action of most-lactamases and is virtually the last effective agent against the vancomycin-resistant Gram-positive bacteria, as well as against multiple antibiotic-resistant Gram-negative organisms. Here, we report the crystal structure, to 1.8 Å resolution, of an acyl-enzyme intermediate for imipenem bound to the TEM-1-lactamase from Escherichia coli, the parent enzyme of 67 clinical variants. The structure indicates an unprecedented conformational change for the complex which accounts for the ability of this antibiotic to resist hydrolytic deactivation by-lactamases. Computational molecular dynamics underscored the importance of the motion of the acyl-enzyme intermediate, which may be a general feature for catalysis by these enzymes. Assuming that the current efforts in the pharmaceutical companies toward discovery and development of novel antibiotics are successful, the use of these new agents would inevitably select for resistance in microbial populations in a relatively short time.

Orotidine-5'-monophosphate decarboxylase (ODCase) has evolved to catalyze a decarboxylation reaction, most probably via a carbanion species at the C6 position of orotidine-5'-monophosphate. We reveal an unusual biochemical pathway of conversion of 6-cyano-uridine-5'-monophosphate by ODCase to barbiturate-5'-monophosphate via perhaps an electrophilic center at the C6 position, leading to inhibition. This potential of ODCase is very useful in the design of novel inhibitors.