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 ε-amino group of a highly conserved lysine residue in acetyl-CoA synthetase (ACS) with a k cat/K m 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.
Iron–sulfur clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some iron–sulfur clusters have ...nonredox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. Here we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe–2S cluster in which one iron ligand is a potentially exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser-491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser-491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg2+ ion 6.5 Å from the 2Fe–2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster and show that Mtb-DHAD is inhibited by a recently discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.
The Mycobacterium tuberculosis enzyme Rv2275 catalyzes the formation of cyclo(L-Tyr-L-Tyr) using two molecules of Tyr-tRNATyr as substrates. The three-dimensional (3D) structure of Rv2275 was ...determined to 2.0-Å resolution, revealing that Rv2275 is structurally related to the class Ic aminoacyl-tRNA synthetase family of enzymes. Mutagenesis and radioactive labeling suggests a covalent intermediate in which L-tyrosine is transferred from Tyr-tRNATyr to an active site serine (Ser88) by transesterification with Glu233 serving as a critical base, catalyzing dipeptide bond formation.
The Gcn5-related
N-acetyltransferases are an enormous superfamily of enzymes that are universally distributed in nature and that use acyl-CoAs to acylate their cognate substrates. In this review, we ...will examine those members of this superfamily that have been both structurally and mechanistically characterized. These include aminoglycoside
N-acetyltransferases, serotonin
N-acetyltransferase, glucosamine-6-phosphate
N-acetyltransferase, the histone acetyltransferases, mycothiol synthase, protein
N-myristoyltransferase, and the Fem family of amino acyl transferases.
Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently ...discovered bifunctional aminoglycoside acetyltransferase (AAC(6′)-Ib variant, AAC(6′)-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6′)-Ib-wt and its bifunctional variant AAC(6′)-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6′)-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 Å. Observation of the direct, and optimally positioned, interaction between the 6′-NH2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6′)-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6′)-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes π-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6′)-Ib-wt and the AAC(6′)-Ib-cr variant involving a ternary complex.
Nicotinamidase/pyrazinamidase (PncA) is involved in the NAD+ salvage pathway of Mycobacterium tuberculosis and other bacteria. In addition to hydrolyzing nicotinamide into nicotinic acid, PncA also ...hydrolyzes the prodrug pyrazinamide to generate the active form of the drug, pyrazinoic acid, which is an essential component of the multidrug treatment of TB. A coupled enzymatic activity assay has been developed for PncA that allows for the spectroscopic observation of enzyme activity. The enzyme activity was essentially pH-independent under the conditions tested; however, the measurement of the pH dependence of iodoacetamide alkylation revealed a pK value of 6.6 for the active site cysteine. Solvent deuterium kinetic isotope effects revealed an inverse value for k cat of 0.64, reconfirming the involvement of a thiol group in the mechanism. A mechanism is proposed for PncA catalysis that is similar to the mechanisms proposed for members of the nitrilase superfamily, in which nucleophilic attack by the active site cysteine generates a tetrahedral intermediate that collapses with the loss of ammonia and subsequent hydrolysis of the thioester bond by water completes the cycle. An inhibitor screen identified the competitive inhibitor 3-pyridine carboxaldehyde with a K i of 290 nM. Additionally, pyrazinecarbonitrile was found to be an irreversible inactivator of PncA, with a k inact/K I of 975 M−1 s−1.
Fluoroquinolones are gaining increasing importance in the treatment of tuberculosis. The expression of MfpA, a member of the pentapeptide repeat family of proteins from Mycobacterium tuberculosis, ...causes resistance to ciprofloxacin and sparfloxacin. This protein binds to DNA gyrase and inhibits its activity. Its three-dimensional structure reveals a fold, which we have named the right-handed quadrilateral {szligbeta} helix, that exhibits size, shape, and electrostatic similarity to B-form DNA. This represents a form of DNA mimicry and explains both its inhibitory effect on DNA gyrase and fluoroquinolone resistance resulting from the protein's expression in vivo.
QnrB1 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.
Tuberculosis (TB) due to
remains a major global infectious disease problem, and a more efficacious vaccine is urgently needed for the control and prevention of disease caused by this organism. We ...previously reported that a genetically modified strain of
called IKEPLUS is a promising TB vaccine candidate. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4
T cell memory, we hypothesized that the specificity of the CD4
T cell response was a critical feature of this protection. Using
assays of interferon gamma production (enzyme-linked immunosorbent spot ELISPOT assays) by splenocytes from IKEPLUS-immunized C57BL/6J mice, we identified an immunogenic peptide within the mycobacterial ribosomal large subunit protein RplJ, encoded by the Rv0651 gene. In a complementary approach, we generated major histocompatibility complex (MHC) class II-restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of these T cell hybridomas against IKEPLUS and ribosomes enriched from IKEPLUS suggested that the CD4
T cell response in IKEPLUS-immunized mice was dominated by the recognition of multiple components of the mycobacterial ribosome. Importantly, CD4
T cells specific for mycobacterial ribosomes accumulate to significant levels in the lungs of IKEPLUS-immunized mice following aerosol challenge with virulent
, consistent with a role for these T cells in protective host immunity in TB. The identification of CD4
T cell responses to defined ribosomal protein epitopes expands the range of antigenic targets for adaptive immune responses to
and may help to inform the design of more effective vaccines against tuberculosis.
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