Structural and functional insights into peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.     

Development of a fluorescence polarization assay for peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Historically, the hydrolyzing activity of Pth has been assayed with radiolabeled N-blocked aminoacyl-tRNAs in assay systems that require the separation of radiolabeled amino acid from the N-blocked aminoacyl-tRNA complex. In the present study, we describe the development of a kinetic fluorescence polarization (FP) assay that enables measurements of Pth activity without the need to separate bound and free tracer. The hydrolyzing activity of Pth was determined by measuring the change in polarization values that resulted from the cleavage of a fluorescently labeled substrate (BODIPY-Lys-tRNA(Lys)). The data were analyzed using an equation describing first-order dissociation and the results showed that the experimental data correlated well with the theoretical curve. A runs test of the residuals showed that the experimental data did not significantly differ from the first-order model. The assay is adaptable to a multiwell format and is sensitive enough to detect Pth-like activity in bacterial cell lysate. The Pth FP assay provides a homogeneous and kinetic format for measuring Pth activity in vitro.     

Metaviromics analysis of marine biofilm reveals a glycoside hydrolase endolysin with high specificity towards Acinetobacter baumannii

Multidrug-resistant (MDR) bacteria are a growing threat to the public health. Among them, the Gram-negative Acinetobacter baumannii is considered today as the most dangerous MDR pathogen. Phage-derived endolysins are peptidoglycan (PG) hydrolytic enzymes that can function as effective tools in the fight against MDR bacteria. In the present work, the viral diversity of a marine environmental sample (biofilm), formed near an industrial zone, was mined for the identification of a putative endolysin (AbLys2) that belongs to the glycoside hydrolase family 24 (GH24, EC 3.2.1.17). The coding sequence of AbLys2 was cloned and expressed in E. coli. The lytic activity and specificity of the recombinant enzyme were evaluated against suspensions of a range of Gram-positive and Gram-negative human pathogens using turbidity assays. AbLys2 displayed enhanced selectivity towards A. baumannii cells, compared to other bacteria. Kinetics analysis was carried out to characterize the dependence of its lytic activity on pH and showed that the enzyme exhibits its maximal activity at pH 5.5. Thermostability analysis showed that AbLys2 displays melting temperature T_m 47.1 °C. Florescence microscopy and cell viability assays established that AbLys2 is active towards live cultures of A. baumannii cells with an inhibitory concentration IC₅₀ 3.41 ± 0.09 μM. Molecular modeling allowed the prediction of important amino acid residues involved in catalysis. The results of the present study suggest that AbLys2 provides efficient lytic and antimicrobial activity towards A. baumannii cells and therefore is a promising new antimicrobial against this pathogen.     

Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and suggests basis for a bifunctional activity

Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-A resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an alpha/beta fold with a four-stranded antiparallel beta-sheet in its core surrounded by two alpha-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.     

Saccharomyces cerevisiae URH1 (Encoding Uridine-Cytidine N-Ribohydrolase): Functional Complementation by a Nucleoside Hydrolase from a Protozoan Parasite and by a Mammalian Uridine Phosphorylase

Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1). Disruption of this gene in a conditionally pyrimidine-auxotrophic S. cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.     

Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-TΨC domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 Å resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies’ results.     

Crystal structure at 1.2 A resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl-tRNAs produced through abortion of translation. This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues. The structure of crystalline peptidyl-tRNA hydrolase could be solved at 1.2 A resolution. It indicates a single alpha/beta globular domain built around a twisted mixed beta-sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica. This similarity allowed the characterization by site-directed mutagenesis of several residues of the active site of peptidyl-tRNA hydrolase. These residues, strictly conserved among the known peptidyl-tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C-end of a neighbouring peptidyl-tRNA hydrolase molecule. Hence, several main chain atoms of three residues belonging to one peptidyl-tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl-tRNA hydrolase molecule. Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction.     

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with cytosine,cytidine, and cytidine diphosphate

The type 1 ribosome inactivating protein from Momordica balsamina (MbRIP1) has been shown to interact with purine bases, adenine and guanine of RNA/DNA. We report here the binding and structural studies of MbRIP1 with a pyrimidine base, cytosine; cytosine containing nucleoside, cytidine; and cytosine containing nucleotide, cytidine diphosphate. All three compounds bound to MbRIP1 at the active site with dissociation constants of 10^?4 M–10^?7 M. As reported earlier, in the structure of native MbRIP1, there are 10 water molecules in the substrate binding site. Upon binding of cytosine to MbRIP1, four water molecules were dislodged from the substrate binding site while five water molecules were dislodged when cytidine bound to MbRIP1. Seven water molecules were dislocated when cytidine diphosphate bound to MbRIP1. This showed that cytidine diphosphate occupied a larger space in the substrate binding site enhancing the buried surface area thus making it a relatively better inhibitor of MbRIP1 as compared to cytosine and cytidine. The key residues involved in the recognition of cytosine, cytidine and cytidine diphosphate were Ile71, Glu85, Tyr111 and Arg163. The orientation of cytosine in the cleft is different from that of adenine or guanine indicating a notable difference in the modes of binding of purine and pyrimidine bases. Since adenine containing nucleosides/nucleotides are suitable substrates, the cytosine containing nucleosides/nucleotides may act as inhibitors.     

Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.

Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.     

Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system.     

Identification of Novel Vaccine Candidates against Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is an emerging opportunistic bacterium associated with nosocomial infections in intensive care units. The alarming increase in infections caused by A. baumannii is strongly associated with enhanced resistance to antibiotics, in particular carbapenems. This, together with the lack of a licensed vaccine, has translated into significant economic, logistic and health impacts to health care facilities. In this study, we combined reverse vaccinology and proteomics to identify surface-exposed and secreted antigens from A. baumannii. Using in silico prediction tools and comparative genome analysis in combination with in vitro proteomic approaches, we identified 42 antigens that could be used as potential vaccine targets. Considering the paucity of effective antibiotics available to treat multidrug-resistant A. baumannii infections, these vaccine targets may serve as a framework for the development of a broadly protective multi-component vaccine, an outcome that would have a major impact on the burden of A. baumannii infections in intensive care units across the globe.     

The chemical synthesis and antibiotic activity of a diverse library of 2-aminobenzimidazole small molecules against MRSA and multidrug-resistant A. baumannii

Multidrug-resistant bacterial infections continue to be a rising global health concern. Herein is described the development of a class of novel 2-aminobenzimidazoles with antibiotic activity. These active 2-aminobenzimidazoles retain their antibiotic activity against several strains of multidrug-resistant Staphylococcus aureus and Acinetobacter baumannii when compared to susceptible strains.     

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.     

Alcohol Enhances Acinetobacter baumannii-Associated Pneumonia and Systemic Dissemination by Impairing Neutrophil Antimicrobial Activity in a Murine Model of Infection

Acinetobacter baumannii (Ab) is a common cause of community-acquired pneumonia (CAP) in chronic alcoholics in tropical and sub-tropical climates and associated with a >50% mortality rate. Using a murine model of alcohol (EtOH) administration, we demonstrated that EtOH enhances Ab-mediated pneumonia leading to systemic infection. Although EtOH did not affect neutrophil recruitment to the lungs of treated mice, it decreased phagocytosis and killing of bacteria by these leukocytes leading to increased microbial burden and severity of disease. Moreover, we determined that mice that received EtOH prior to Ab infection were immunologically impaired, which was reflected in increased pulmonary inflammation, sequential dissemination to the liver and kidneys, and decreased survival. Furthermore, immunosuppression by EtOH was associated with deregulation of cytokine production in the organs of infected mice. This study establishes that EtOH impairs immunity in vivo exacerbating Ab infection and disease progression. The ability of Ab to cause disease in alcoholics warrants the study of its virulence mechanisms and host interactions.     

Genome Sequence of Acinetobacter baumannii TYTH-1

Acinetobacter baumannii has emerged recently as a major cause of health care-associated infections due to the extent of its antimicrobial resistance and its propensity to cause large nosocomial outbreaks. Here we report the genome sequence of Acinetobacter baumannii TYTH-1 isolated in Taiwan during 2008.     

Structural and binding studies of phosphopantetheine adenylyl transferase from Acinetobacter baumannii

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4′-phosphopantetheine (pPant) yielding 3′- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway_. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri‑sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10⁻⁸ M and 10⁻⁵ M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Å resolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.     

A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli

The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth^ts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth^ts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth^ts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.     

Occurrence of an Environmental Acinetobacter baumannii Strain Similar to a Clinical Isolate in Paleosol from Croatia

Over the past decade, bacteria of the genus Acinetobacter have emerged as a leading cause of hospital-acquired infections. Outbreaks of Acinetobacter infections are considered to be caused exclusively by contamination and transmission in hospital environments. The natural habitats of clinically important multiresistant Acinetobacter spp. remain to be defined. In this paper, we report an incidental finding of a viable multidrug-resistant strain of Acinetobacter baumannii, related to clinical isolates, in acid paleosol from Croatia. The environmental isolate of A. baumannii showed 87% similarity to a clinical isolate originating from a hospital in this geographic area and was resistant to gentamicin, trimethoprim-sulfamethoxazole, ciprofloxacin, and levofloxacin. In paleosol, the isolate was able to survive a low pH (3.37), desiccation, and a high temperature (50°C). The probable source of A. baumannii in paleosol is illegally disposed waste of external origin situated in the abandoned quarry near the sampling site. The bacteria could have been leached from waste by storm water and thus infiltrated the paleosol.