NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism.

The specter of a return to an era in which infectious disease looms as a significant threat to human health is not just hyperbole; there are serious concerns about the widespread overuse and misuse of antibiotics contributing to increased antibiotic resistance in pathogens. The recent discovery of a new enzyme, first identified in Klebsiella pneumoniae from a patient from New Delhi and denoted as NDM-1, represents an example of extreme promiscuity: It hydrolyzes and inactivates nearly all known ß-lactam-based antibiotics with startling efficiency. NDM-1 can utilize different metal cofactors and seems to exploit an alternative mechanism based on the reaction conditions. Here we report the results of a combined experimental and theoretical study that examines the substrate, metal binding, and catalytic mechanism of the enzyme. We utilize structures obtained through X-ray crystallography, biochemical assays, and numerical simulation to construct a model of the enzyme catalytic pathway. The NDM-1 enzyme interacts with the substrate solely through zinc, or other metals, bound in the active site, explaining the observed lack of specificity against a broad range of ß-lactam antibiotic agents. The zinc ions also serve to activate a water molecule that hydrolyzes the ß-lactam ring through a proton shuttle.

Characterization of member of DUF1888 protein family, self-cleaving and self-assembling endopeptidase.

The crystal structure of SO1698 protein from Shewanella oneidensis was determined by a SAD method and refined to 1.57 Å. The structure is a ß sandwich that unexpectedly consists of two polypeptides; the N-terminal fragment includes residues 1-116, and the C-terminal one includes residues 117-125. Electron density also displayed the Lys-98 side chain covalently linked to Asp-116. The putative active site residues involved in self-cleavage were identified; point mutants were produced and characterized structurally and in a biochemical assay. Numerical simulations utilizing molecular dynamics and hybrid quantum/classical calculations suggest a mechanism involving activation of a water molecule coordinated by a catalytic aspartic acid.

Cervical spinal cord injury and the need for cardiovascular intervention.

HYPOTHESIS: The level of cervical spinal cord injury (CSCI) can be used to predict the need for a cardiovascular intervention. DESIGN: Retrospective review. Data included level of spinal cord injury, Injury Severity Score, lowest heart rate, and systolic blood pressure in the first 24 hours and intensive care unit course. The level of CSCI was divided into high (cord level C1-C5) or low (cord level C6-C7). Neurogenic shock was defined as bradycardia with hypotension. Statistical analysis was performed with the t test and the chi2 test. SETTING: Level I trauma center. PATIENTS: The patients studied were those with quadriplegia who experienced a CSCI and were admitted to the hospital between December 1, 1993, and October 31, 2001. INTERVENTIONS: Pressors, chronotropic agents, and pacemakers.Main Outcome Measure Use of a cardiovascular intervention in the presence of neurogenic shock. RESULTS: Eighty-three patients met the criteria for CSCI and quadriplegia, 62 in the high (C1-C5) and 21 in the low (C6-C7) level. There was no significant difference between the 2 groups in mean +/- SD age (38.2+/-17.8 vs 34.7+/-15.6 years; P=.43), mean +/- SD Injury Severity Score (35.7+/-17.5 vs 32.5+/-11.2; P=.44), mean +/- SD admission base deficit (-0.7+/-3.6 vs 0.7+/-2.7; P=.06), or mortality (12 [19%] of 62 patients vs 2 [10%] of 21 patients; P=.29). Neurogenic shock was present in 19 (31%) of the 62 patients with high CSCI and in 5 (24%) of the 21 patients with low CSCI (P=.56). There was a marked difference in the use of a cardiovascular intervention between those with a high and those with a low CSCI: 15 (24%) of 62 patients vs 1 (5%) of 21 patients (P=.02). Two patients with C1 through C5 spinal cord injuries required cardiac pacemakers. CONCLUSIONS: There was no significant difference in the frequency of neurogenic shock by injury level. Patients with a high CSCI (C1-C5) had a significantly greater requirement for a cardiovascular intervention compared with patients with lower injuries (C6-C7).

Dynamical probing of allosteric control in nuclear receptors.

The dynamical behavior of the nuclear receptor LXR/RXR heterodimer was investigated with molecular dynamics simulations. The simulations reveal correlated motion between residues across the dimer interface that depends significantly on occupation of the ligand binding sites of the monomers. These results are broadly consistent with the observed experimental behavior of the dimers, where structural perturbation is thought to be a key element in signal transduction. Our results provide dynamical support for this model of allosteric control.

High-resolution structure of the nitrile reductase QueF combined with molecular simulations provide insight into enzyme mechanism.

Here, we report the 1.53-Å crystal structure of the enzyme 7-cyano-7-deazaguanine reductase (QueF) from Vibrio cholerae, which is responsible for the complete reduction of a nitrile (CN) bond to a primary amine (H(2)C-NH(2)). At present, this is the only example of a biological pathway that includes reduction of a nitrile bond, establishing QueF as particularly noteworthy. The structure of the QueF monomer resembles two connected ferrodoxin-like domains that assemble into dimers. Ligands identified in the crystal structure suggest the likely binding conformation of the native substrates NADPH and 7-cyano-7-deazaguanine. We also report on a series of numerical simulations that have shed light on the mechanism by which this enzyme affects the transfer of four protons (and electrons) to the 7-cyano-7-deazaguanine substrate. In particular, the simulations suggest that the initial step of the catalytic process is the formation of a covalent adduct with the residue Cys194, in agreement with previous studies. The crystal structure also suggests that two conserved residues (His233 and Asp102) play an important role in the delivery of a fourth proton to the substrate.