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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.

The article explains various mechanisms that are involved in the catalysis and inhibition of nicotinamidase/pyrazinamidase (PncA) from Mycobacterium tuberculosis. Pyrazinecarbonitrile is shown to be one compound that acts as an irreversible inactivator of PcnA.

The kinetic characterization of bifunctional aminoglycoside acetyltransferase-Ib variant (AAC(6')-Ib-cr) and its wild-type parent enzyme along with the three-dimensional structure of the wild-type enzyme is described. It highlights the adaptive potential of AACs driven by the selective pressures of fluoroquinolone use.

The studies on the basis of kinetic properties, pH dependence, and structural information of SSAT in Escherichia coli reveal an acid/base-assisted reaction catalyzed by spermidine/spermine acetyltransferase (SSAT), involving a ternary complex.

The three-dimensional structure of the first member of the pentapeptide repeat protein (PRP) family is determined for the fluoroquinolene resistance protein (MfpA) from Mycobacterium tuberculosis. The structure shows that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix, MfpA binds to DNA gyrase and inhibits its activity and the rod-shaped, dimeric protein displays remarkable similarity in size, shape and electrostatics to DNA.

QnrBl is a plasmid-encoded pentapeptide repeat protein (PRP) that confers a moderate degree of resistance to fluoroquinolones. Its gene was cloned into an expression vector with an N-terminal polyhistidine tag, and the protein was purified by nickel affinity chromatography. The structure of QnrB1 was determined by a combination of trypsinolysis, surface mutagenesis, and single anomalous dispersion phasing. QnrB1 folds as a right-handed quadrilateral β-helix with a highly asymmetric dimeric structure typical of PRP-topoisomerase poison resistance factors. The threading of pentapeptides into the β-helical fold is interrupted by two noncanonical PRP sequences that produce outward projecting loops that interrupt the regularity of the PRP surface. Deletion of the larger upper loop eliminated the protective effect of QnrB1 on DNA gyrase toward inhibition by quinolones, whereas deletion of the smaller lower loop drastically reduced the protective effect. These loops are conserved among all plasmid-based Qnr variants (QnrA, QnrC, QnrD, and QnrS) and some chromosomally encoded Qnr varieties. A mechanism in which PRP-topoisomerase poison resistance factors bind to and disrupt the quinolone-DNA-gyrase interaction is proposed. [ABSTRACT FROM AUTHOR]

Recent proteomics studies have revealed that protein acetylation is an abundant and evolutionarily conserved post-translational modification from prokaryotes to eukaryotes. Although an astonishing number of acetylated proteins have been identified in those studies, the acetyltransferases that target these proteins remain largely unknown. Here we characterized MSMEG_5458, one of the GCN5-related N-acetyltransferases (GNAT's) in Mycobacterium smegmatis, and show that it is a protein acetyltransferase (MsPat) that specifically acetylates the e-amino group of a highly conserved lysine residue in acetyl-CoA synthetase (ACS) with a kcat/Km of nearly 104 M-1 s-1. This acetylation results in the inactivation of ACS activity. Lysine acetylation by MsPat is dependent on 3′,5′-cyclic adenosine monophosphate (cAMP), an important second messenger, indicating that MsPat is a downstream target of the intracellular cAMP signaling pathway. To the best of our knowledge, this is the first protein acetyltransferase in mycobacteria that both is dependent on cAMP and targets a central metabolic enzyme by a specific post-translational modification. Since cAMP is synthesized by adenylate cyclases (AC's) that sense various environmental signals, we hypothesize that the acetylation and inactivation of ACS is important for mycobacteria to adjust to environmental changes. In addition, we show that Rv1151c, a sirtuin-like deacetylase in Mycobacterium tuberculosis, reactivates acetylated ACS through an NAD+-dependent deacetylation. Therefore, Pat and the sirtuin-like deacetylase in mycobacteria constitute a reversible acetylation system that regulates the activity of ACS. [ABSTRACT FROM AUTHOR]

The N¹-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N¹-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 Å resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex. [ABSTRACT FROM AUTHOR]

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 Å, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral β-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein. [ABSTRACT FROM AUTHOR]

Presents a kinetic and mechanistic characterization of recombinant Lactobacillus viridescens FemX (LvFemX), UDP-N-acetylmuramoyl pentapeptide-lysine N[sup6]-alanyltransferase. Substrate specificity of LvFemX; Modification of carboxyl groups and site-directed mutagenesis; Mechanism of the enzyme; Involvement of the carboxyl groups in the catalytic function of the enzyme.

Investigates the thermodynamics of aminoglycoside and acyl-coenzyme A binding to the Salmonella enterica. Administration of aminoglycoside antibiotics in bacterial infections; Suppression of bacterial protein synthesis; Mechanism of bacterial resistance to aminoglycosides.

The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6× His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC(50)) of 1.2 μM, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-Å resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria.

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 A, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral beta-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein.