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We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxy-butyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.

The characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV is reported. The pH dependence of the kinetic parameters analysis reveal acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding.

The Mycobacterium tuberculosis gene Rv2747 encodes a novel 19-kDa ArgA that catalyzes the initial step in L-arginiue biosynthesis, namely the conversion of L-glutamate to [alpha]-N-acetyl-e-glutamate. Initial velocity studies reveal that Rv2747 proceeds through a sequential kinetic mechanism, with [K.sub.m] values of 280 mM for L-glutamine and 150 [micro]M for acetyl-coenzyme A and with a [k.sub.cat] value of 200 [min.sup.-1]. Initial velocity studies with L-glutamate showed that even at concentrations of 600 mM, saturation was not observed. Therefore, only a [k.sub.cat]/[K.sub.m] value of 125 [M.sup.-1] [min.sup.-1] can be calculated. Inhibition studies reveal that the enzyme is strongly regulated by L-arginine, the end product of the pathway (50% inhibitory concentration, 26 [micro]M). The enzyme was completely inhibited by 500 [micro]M arginine, with a Hill coefficient of 0.60, indicating negatively cooperative binding of L-arginine.

The functional roles for His44, His47, Asn69, Gln72, Lys160 and Gln164 residues in the binding of substrates and the pantoyl adenylate intermediate are evaluated by constructing six Ala mutants and determining their catalytic properties. The results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residues are necessary for the formation and stabilization of pantoyl adenylate intermediate in the pantothenate synthetase reaction.

Research has been conducted on ketopantoate hydroxymethyltransferase. The cloning and expression of the Mycobacterium tuberculosis gene encoding this hydroxymethyltransferase has been carrried out, and the rate of tetrahydrofolate-independent hydroxymethyltransferase chemistry between formaldehyde and alpha-ketoisovalerate has been measured via the use of NMR spectroscopy.

The multi-phasic time course reactions of Mycobacterium tuberculosis lipoamide dehydrogenase reveals the formation of a transient reduced flavin intermediate. The compound is highly unstable.

The authors presents the topics which are going to be addressed at the national meeting of the American Chemical Society, August 18-22. These topics include comparative genomics, proteins engineered by molecular evolution, small molecule protein kinase inhibitors and glycosaminoglycan oligosaccharides' chemoenzymatic synthesis.

Research has been conducted on the chromosomally encoded aminoglycoside AAC(6')-Iy from Salmonella enterica. The study of the thermodynamic parameters and equilibrium binding of the acyl-coenzyme A derivatives to AAC(6')-Iy and aminoglycosides, investigated via the use of the fluorescence spectroscopy and isothermal titration calorimetry, is discussed.

Results show that the kinetic mechanism of the pantothenate synthetase from Mycobacterium tuberculosis is Bi Uni Uni Bi Ping Pong with successive ATP and D-pantoate binding, release of pyrophosphate, and binding of beta-alanine and release of pantothenate and AMP.

Research describes the cloning and expression in Escherichia coli the gene Rv0462 encoding dihydrolipoamide dehydrogenase of Mycobacterium tuberculosis. The gene Rv0462 is termed lpdC to differentiate it from lpdA or lpdB. Physicochemical and steady-state kinetic mechanism of the enzyme are presented.

Research has been conducted on the purine nucleoside phosphorylases (PNPs) encoded by Mycobacterium tuberculosis. The crystal structure of M. tuberculosis PNP has been investigated in complex with the transition-state analogue immucillin-H and inorganic phosphate to confirm its trimeric structure.

Research has been conducted on the effect of Mycobacterium tuberculosis genome sequence on purine salvage pathways. The results indicate that M. tuberculosis deoD gene exhibit purine nucleoside activity after having been cloned, expressed and purified.

Research reveals that His444-Glu449 ion pair and Cys39 residues are involved in the disulfide reduction mechanism catalyzed by the enzyme mycothione reductase. Data indicate that the enzyme kinetics are influenced by pH suggesting a flavoprotein disulfide mechanism in which the oxidative half-reaction is faster than the reductive half-reaction.

Research has been conducted on the chromosomally encoded aminoglycoside N-acetyltransferase from Salmonella enterica that resists toward aminoglycoside antibiotics. The cloning and expression of the structural gene demonstrated that the purified enzyme exists in solution as a dimer of ca. 17 000 Da monomers.

Results reveal that amino acid residues Lys176 and Glu256 are at the active site of the Escherichia coli ketopantoate reductase mediating ketopantoate binding and catalysis. Substitution of alanine residue to either of the active site residues results in a marked reduction in the catalytic efficiency of the enzyme.

A kinetic study of phenylalanine dehydrogenase shows that NAD+ binds before phenylalanine, and ammonia is released first, then phenylpyruvate, and then NADH. Lys78 and Asp118 are the catalytic residues at the active site.

Homoserine transacetylase has been cloned from Homophilus influenzae and a detailed enzymatic study made of the enzyme. Direct evidence of formation of an acetyl-enzyme intermediate has been found using rapid-quench labeling studies. A chemical mechanism is proposed for the important member of the acyltransferase family. Its mechanism is contrasted with that of homoserine transuccinylase.

Research has been conducted on the ketopantoate reductase. The catalysis of the NADPH-dependent reduction of alpha-ketopantoate is described.

Research has been conducted on the Escherichia coli argE-encoded N-acetyl-L-ornithine deacetylase. The cloning, expression and purification of the deacetylase is described.

Enzyme-catalyzed acylation of homoserine is discussed relative to characterization, mechanistically, of the Esherichia coli metA-encoded homoserine transsuccinylase. Kinetic parameters for natural substrates have been found, and alternative substrates have been found. A ping-pong kinetic mechanism is suggested. An inverse isotope effect on the reverse reaction is indicated in solvent kinetic isotope effect studies. No effect is indicated on the forward reaction. A model of the enzymatic chemical mechanism is proposed.

The cloning, expression, purification and characterization of Mycobacterium tuberculosis mycothione reductase were conducted. The mtr-encoded mycothione reductase is a homodimeric flavoprotein containing FAD as a cofactor. The enzyme displays a bi-bi- ping-pong kinetic mechanism and possesses both transhydrogenase and diaphorase activities. The reductive titration of mycothione reductase suggests the presence of a charge-transfer complex of a thiolate and FAD in the two-electron reduced enzyme.

A study was conducted to analyze the chemical mechanism of Haemophilus influenzae diaminopimelate epimerase, a member of the family of PLP-independent amino acid racemases. Primary deuterium and solvent kinetic isotope effects were obtained through spectropolarimeter measurements. Results indicated that D,L-meso-diaminopimelate epimerase supports a number of novel characteristics not seen with other PLP-independent racemases.

Escherichia coli argD-encoded N-acetylornithine aminotransferase (NAcOATase) exhibits catalytic competence in both NAcOATase and alpha-ketoglutarate aminotransferase activity. This capability suggests that the enzyme might be capable of functioning in two important amino acid biosynthetic routes, making it an ideal candidate for inhibitor design. This ability may also be the explanation behind the failure to identify the location of the dapC gene.

A study was conducted to synthesize, express, purify, crystallize and determine the three-dimensional structure of the Haemophilus influenzae diaminopimelate epimerase to 2.7 Angstrom. Results indicate that this epimerase is a monomeric, 274-amino acid enzyme with two structurally homologous domains. This enzyme is of overall cylindrical shape and measures roughly 53 by 23 by 25 Angstrom. Other findings in the experiment are thoroughly discussed.

Research was conducted to investigate the hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase. Metal activation, solvent isotope effects, and kinetic mechanism studies were performed. Results indicate that the desuccinylase is a metalloenzyme, as are a number of enzymes which catalyze amide bond hydrolysis. A mechanism for the desuccinylase, which is similar to those proposed for other metal-dependent amidases, is proposed.

Research was conducted to examine the conformational change and active site structure of tetrahydrodipicolinate N-succinyltransferase. The crystal structures of two ternary complexes of the enzyme formed with L2AP/CoA and L2A6OP/CoA are described. The structures define the active site location and conformation of the bound amino acid and cofactor. Results indicate that the conformational change is directly connected to interactions between the bound amino acid substrate and cofactor and the enzyme.

The structure of the binary enzyme meso-diaminopimelate was resolved and refined against 2.2 A x-ray diffraction data. Corynebacterium glutamicum diaminopimelate dehydrogenase was crystallized at room temperature and collected by hanging drop vapor diffusion. The enzyme consisted of three domains, dinucleotide binding domain, dimerization domain and substrate-binding domain, bound in an elongated cavity with a single orientation for binding substrates so that the D-amino acid center is positioned near the oxidized nucleotide.

The three-dimensional structure of Escherichia coli dihydrodipicolinate reductase in complex with NADH and the inhibitor 2,6-pyridinedicarboxylate was solved using combined molecular replacement and noncrystallographic symmetry averaging procedures and refined against 2.6 angstrom resolution data to a crystallographic R-factor of 21.4%. Residues involved in binding of the inhibitor and the residues involved in catalysis were identified based on the three-dimensional structure. A proposed chemical mechanism for the enzyme is presented.

Hydrogen/deuterium exchange and electrospray ionization mass spectrometry were utilized to determine the binding of Escherichia coli dihydrodipicolinate reductase to reduced nicotinamide adenine dinucleotide phosphate (NADPH). The dihydrodipicolinate reductase-NADPH (DHPR-NADPH) complex was composed of a two-domain protein with four protein molecules that exhibited reduced deuterium exchange due to the binding of the substrate or inhibitor. Furthermore, pepsin digestion of the DHPR-NADPH complex indicated the role two additional peptides as specific regions for the binding of NADH.

The X-ray crystal structure of tetrahydrodipicolinate N-succinyltransferase shows that the enzyme is trimeric. Each subunit has tandem repeats of hexapeptide amino acid sequence motif which forms left-handed parallel beta-helix (L-beta-H). Each L-beta-H from the three subunits is parallel to the three-fold axis of the trimeric enzyme. The binding sites of different inhibitors of the enzyme indicate that the active site is located at the junction between the subunits of the trimer.

The crystal structure of the diaminopimelate dehydrogenase-NADP+ complex from Corynebacterium glutamicum was determined by the multiple isomorphous replacement method at 2.2 angstroms resolution. Crystallography data revealed that the enzyme is a homodimer comprised of unidentical subunits, with each subunit consisting of three domains. The N-terminal domain contained six central beta-strands surrounded by five alpha-helices; the dimerization domain contained two alpha-helices and three beta-strands, and forms the monomer-monomer interface of the dimer; and the C-terminal domain which consists od six beta-strands and five alpha-helices. The N-terminal and C-terminal domains were suggested to contain the enzyme's binding and active sites.

The thermodynamic parameters of binding and the three-dimensional structures of the corresponding enzyme-nucleotide complexes are determined by isothermal titration calorimetry to study the interactions which cause the unique nucleotide specificity of Escherichia coli dihydropicolinate reductase. The 2'-phosphate of NADPH interacts electrostatically with Arg39, while in the NADH complex this interaction is replaced by hydrogen bonds between the 2' and 3' adenosyl ribose hydroxyls and Glu38.

The pyrophosphate analog phosphonoacetate was used to examine kinetic isotope effects on the phosphorolysis of orotidine 5'-monophosphate (OMP) catalyzed by Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) to produce orotate and alpha-D-phosphoribosylpyrophosphate. The results show that the transition state of OMP is similar to a classical oxycarbonium ion with the leaving orotate base having substantial buildup of negative charge. A model of the OMP transition state in the OPRTase active site and a chemical mechanism to explain the results are presented.

The Lys-Glu (K60-E201) base pair, which occurs in the enzyme trypanothione reductase in many species of Trypanosoma, plays a regulatory role in the redox potential and reactivity of the enzyme. Mutants in which the K60 is replaced by arginine produce an enzyme incapable of catalytic activity and replacement of the E201 residue with glutamine or aspartate produces an enzyme with reduced activity. Trypanothione reductase is a flavoprotein that plays a role in protection against oxidative stress and helps in reducing the levels of hydrogen peroxide in the organism.

The inhA gene makes a major protein target of ethioinamide and isoniazid action in Mycobacterium tuberculosis and antibacterial compound involved in fatty acid biosynthesis. The NADH-specific reduction of 2-trans-enoyl-ACP necessary for fatty acid synthesis is catalyzed by the Mycobacterium tuberculosis InhA protein. Steady-state kinetics analysis shows that the two long chain substrates attach to InhA through a sequential kinetic process.