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The loop comprising residues 112-121 of the Enterobacter cloacae enzyme MurA undergoes several structural changes upon binding its substrate. The loop contains a hydrogen-bonding network that stabilizes the conformation in the unliganded state.

The crystal structure and kinetic characterization of enzyme MurA from Enterobacter cloacae and Escherichia coli is described. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflected the importance of noncovalent binding potential, even for covalent inhibitors for ensuring inactivation efficiency and specificity.

Combination X-ray crystallography and solution NMR spectroscopy were used to characterize the ferredoxin nicotinamide adenine dinucleotide phosphate reductase from Pseudomonas aeruginosa (pa-FPR) in complex with [NADP.sup.+]. The observations from NMR spectra of pa-FPR and the pa-FPR-NADP complex provided insights into the C'-terminal sequence residues, which does not undergo conformational exchange in the presence or absence of [NADP.sup.+] and the stepwise-electron-proton transfer of hydride in the oxidation of NADPH by FPR enzymes.

The differential inhibition of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzymes by analogues of tetrahedral reaction intermediate (TI) is examined. Results reveal that class II EPSPS are generally less susceptible to inhibition by these TI analogues.

The products of the two genes encoding electron transfer proteins upregulated by Pseudomonas aeruginosa in response to iron starvation are identified as the redox partners of the heme oxygenase expressed under low-iron stress conditions.

Calpain is ubiquitous Ca(super 2+) activated proteolytic enzyme, where a crystal structure of the human mu-calpain protease core (hmuI-II) containing a Gly213Ala mutation and covalently inactivated by a peptidomimetic (ZLLYCH2F). The hydrogen bonding and hydrophobic interaction between active site residues and the inhibitor where the terminal ZL portion of the inhibitor is solvent based in recognition of calpain's natural protein.

The high-resolution structures of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibited by (R)- and (S)-configured phosphonate analogues of the tetrahedral reaction intermediate are determined. The results suggest that the structural events during open-closed transition of EPSPS are changed as a result of inhibitor action.

Cyclin-dependent kinases (CDKs) areserine/threonine protein kinasesthat act as key regulatory elements in cell cycle progression. Wedescribe the development of highly potent diaminothiazole inhibitorsof CDK2 (IC50= 0.0009–0.0015 μM) from a singlehit compound with weak inhibitory activity (IC50= 15 μM),discovered by high-throughput screening. Structure-based design wasperformed using 35 cocrystal structures of CDK2 liganded with distinctanalogues of the parent compound. The profiling of compound 51against a panel of 339 kinases revealed high selectivityfor CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4,and CDK6. Compound 51inhibited the proliferation of13 out of 15 cancer cell lines with IC50values between0.27 and 6.9 μM, which correlated with the complete suppressionof retinoblastoma phosphorylation and the onset of apoptosis. Combined,the results demonstrate the potential of this new inhibitors seriesfor further development into CDK-specific chemical probes or therapeutics. [ABSTRACT FROM AUTHOR]

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68 Ga-DOTA-TOC, to produce the dual-labeled analog, 68 Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68 Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68 Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68 Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, O2. Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band (gamma(max) 600 nm) and lack of fluorescence, making it easily distinguished from the subsequently formed inactivated enzyme, which has no long wavelength absorption (gamma(max) 318, 368 nm) and which is strongly fluorescent. Inactivation is also accomplished by reaction of the reduced enzyme with 2-keto-3-butynoate. The absorbance and fluorescence characteristics of the inactivated enzyme are similar to those of a model compound, C(4a), N(5)-propano-bridged FMN bound to apolactate oxidase. That the modified chromophore of the inactivated enzyme is an adduct involving both the C(4a) and N5 positions is further supported by the spectral and fluorescence changes resulting from treatment of the inactivated enzyme with borohydride.

2-Hydroxy-3-butynoic acid is a suicide substrate for Mycobacterium smegmatis lactate oxidase. Inactivation occurs by covalent modification of enzyme-bound FMN and does not involve labeling of the apoprotein. The spectrum of the enzyme bound adduct suggests that it is a 4a, 5-dihydroflavin derivative. When this adduct is released from the enzyme, a complex mixture of unstable compounds is obtained. When the initially formed enzyme-bound adduct is reduced with NaBH4, a major stable species can be resolved from the enzyme and can be isolated and purified. The structure was established by appropriate isotope substitutions. Fourier transform NMR spectroscopy, chemical reactivity, and synthesis of a model compound. The structure of the isolated adduct is structure II, Scheme II. The structure proposed for the adduct initially formed on the enzyme is structure VII, Scheme II.