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The C-terminal domain of the Salmonella virulence factor SseI is structurally similar to the cysteine protease superfamily and contains the conserved catalytic triad characteristic of members of this family., SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells.

We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation (Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A.103 ,1254 -125916432219). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.

Many animal models of bacterial diseases are hampered by differences in tissue tropism and the course of pathogenesis. In a recent issue of Cell , by rationally mutating a surface invasion protein (InlA) to have higher binding affinity for its cognate host receptor (E-cadherin), Wollert et al. were able to "murinize" Listeria monocytogenes , creating a strain capable of invading intestinal epithelial cells in mice, mimicking the route of infection in humans.

We present the discovery and optimization of a novel series of inhibitors of bacterial UDP-N-acetylglucosamine 2-epimerase (called 2-epimerase in this paper). Starting from virtual screening hits, the activity of various inhibitory molecules was optimized using a combination of structure-based and rational design approaches. We successfully designed and identified a 2-epimerase inhibitor (compound 12-ES-Na, that we named Epimerox) which blocked the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.9 μM MIC (minimum inhibitory concentration) and showed potent broad-range activity against all Gram-positive bacteria that were tested. Additionally a microplate coupled assay was performed to further confirm that the 2-epimerase inhibition of Epimerox was through a target-specific mechanism. Furthermore, Epimerox demonstrated in vivo efficacy and had a pharmacokinetic profile that is consonant with it being developed into a promising new antibiotic agent for treatment of infections caused by Gram-positive bacteria.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (

A previously undescribed spectrokinetic assay for the entry of water into the distal heme pocket of wild-type and mutant myoglobins is presented. Nanosecond photolysis difference spectra were measured in the visible bands of sperm whale myoglobin as a function of distal pocket mutation and temperature. A small blue shift in the 560-nm deoxy absorption peak marked water entry several hundred nanoseconds after CO photodissociation. The observed rate suggests that water entry is rate-limited by the escape of internal dissociated CO. The heme pocket hydration and geminate recombination yields were found to be the primary factors controlling the overall bimolecular association rate constants for CO binding to the mutants studied. The kinetic analysis provides estimates of 84%, 60%, 40%, 0%, and 99% for the steady-state hydrations of wild-type, H64Q, H64A, H64L, and V68F deoxymyoglobin, respectively. The second-order rate constants for CO and H 2 O entry into the empty distal pocket of myoglobin are markedly different, 8 × 10 7 and 2 × 10 5 M –1 ·s –1 , respectively, suggesting that hydrophobic partitioning of the apolar gas from the aqueous phase into the relatively apolar protein interior lowers the free energy barrier for CO entry.