The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Identification of potential targets in Staphylococcus aureus N315 using computer aided protein data analysis

Staphylococcus aureus is a gram positive bacterium, responsible for both community-acquired and hospital-acquired infection, resulting in a mortality rate of 39%. 43.2% resistance to methicilin and emerging resistance to Fluroquinolone and Oxazolidinone, have evoked the necessity of the establishment of alternative and effective therapeutic approach to treat this bacteria. In this computational study, various database and online software are used to determine some specific targets of Staphylococcus aureus N315 other than those used by Penicillin, Quinolone and Oxazolidinone. For this purpose, among 302 essential proteins, 101 nonhomologous proteins were accrued and 64 proteins which are unique in several metabolic pathways of S. aureus were isolated by using metabolic pathway analysis tools. Furthermore, 7 essentially unique enzymes involved in exclusive metabolic pathways were revealed by this research, which can be potential drug target. Along with these important enzymes, 15 non-homologous proteins located on membrane were identified, which can play a vital role as potential therapeutic targets for the future researchers.     

The Structure of Sedoheptulose-7-Phosphate Isomerase from Burkholderia pseudomallei Reveals a Zinc Binding Site at the Heart of the Active Site

Heptoses are found in the surface polysaccharides of most bacteria, contributing to structures that are essential for virulence and antibiotic resistance. Consequently, the biosynthetic enzymes for these sugars are attractive targets for novel antibiotics. The best characterized biosynthetic enzyme is GmhA, which catalyzes the conversion of sedoheptulose-7-phosphate into d-glycero-d-manno-heptopyranose-7-phosphate, the first step in the biosynthesis of heptose. Here, the structure of GmhA from Burkholderia pseudomallei is reported. This enzyme contains a zinc ion at the heart of its active site: this ion stabilizes the active, closed form of the enzyme and presents coordinating side chains as a potential acid and base to drive catalysis. A complex with the product demonstrates that the enzyme retains activity in the crystal and thus suggests that the closed conformation is catalytically relevant and is an excellent target for the development of therapeutics. A revised mechanism for the action of GmhA is postulated on the basis of this structure and the activity of B. pseudomallei GmhA mutants.     

Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2′) from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2′). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2′) is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.     

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using ³¹P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.     

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-d-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.     

A comparative study of class 1 integrons in Acinetobacter baumannii

Multidrug resistance (MDR) in Acinetobacter baumannii is increasingly reported and has become a significant public concern. The method responsible for the acquisition of resistance genes via integrons from the environment or intra-species in A. baumannii remains to be understood. This study was performed to investigate the transmission route of these integrons using a comparative analysis of published A. baumannii complete genomes. The phylogenetic analysis of A. baumannii type 1 integrases (IntI1) showed that the integrons could be transferred across the two evolutionary lineages, the international clone I (IC I) and clone II (IC II) strains. In addition, the integrons in A. baumannii strains were mainly responsible for the transfer of resistance genes for two types of long-term usage antibiotics and antiseptics, such as aminoglycosides, chloramphenicol and the quaternary-ammonium-compound family. The in silico comparative analysis of known integron integrases revealed that the intI genes were phylogenetically related among A. baumannii strains and some microorganisms living in a sediment community, implicating that the integrons of A. baumannii might have originated from those microorganisms belonging to the β-preoteobacterial class in the sediment environment. The data suggest that the gain of class 1 integrons in A. baumannii strains may have started before the antibiotic era. This report shows that the origins of A. baumannii class 1 integrons may be the soil environment and that the resistance genes included in integrons are horizontally transferred across all the A. baumannii genomes, including IC I and IC II.     

Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.     

鲍曼不动杆菌碳青霉烯耐药性与生物膜形成及O-甘露糖蛋白相关性研究

鲍曼不动杆菌(Acinetobacter baumannii)是引起医院感染的常见致病菌,该细菌不仅容易产生耐药性,而且在人体及无生命物质表面易形成生物膜,临床治疗较为棘手。从临床分离24株鲍曼不动杆菌,药物敏感试验观察这些分离株对常用抗菌药物的敏感性,针对耐碳青霉烯鲍曼不动杆菌,检测是否含有耐药基因碳青霉烯酶基因OXA-23,采用结晶紫染色法观察耐药性与生物膜形成的相关性,并用刀豆蛋白凝集素结合试验及质谱分析耐药性与O-甘露糖蛋白的相关性。结果显示鲍曼不动杆菌耐药性与生物膜形成呈正相关,某些O-甘露糖蛋白表达有利于细菌获得耐药性。     

Discovery of a New Genetic Variant of Methionine Aminopeptidase from Streptococci with Possible Post-Translational Modifications: Biochemical and Structural Characterization

Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the ‘insert one’. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M. tuberculosis and human Type I MetAPs as well as the 60 amino acid insert in the human Type II enzyme suggesting possible common function. In addition, one of the β-hairpins within in the catalytic domain undergoes a flip placing a residue which is essential for enzyme activity away from the active site and the β-hairpin loop of this secondary structure in the active site obstructing substrate binding. This is the first example of a MetAP crystallizing in the inactive form.     

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.     

Conservation of structure and mechanism by Trm5 enzymes

Enzymes of the Trm5 family catalyze methyl transfer from S-adenosyl methionine (AdoMet) to the N¹ of G37 to synthesize m¹G37-tRNA as a critical determinant to prevent ribosome frameshift errors. Trm5 is specific to eukaryotes and archaea, and it is unrelated in evolution from the bacterial counterpart TrmD, which is a leading anti-bacterial target. The successful targeting of TrmD requires detailed information on Trm5 to avoid cross-species inhibition. However, most information on Trm5 is derived from studies of the archaeal enzyme Methanococcus jannaschii (MjTrm5), whereas little information is available for eukaryotic enzymes. Here we use human Trm5 (Homo sapiens; HsTrm5) as an example of eukaryotic enzymes and demonstrate that it has retained key features of catalytic properties of the archaeal MjTrm5, including the involvement of a general base to mediate one proton transfer. We also address the protease sensitivity of the human enzyme upon expression in bacteria. Using the tRNA-bound crystal structure of the archaeal enzyme as a model, we have identified a single substitution in the human enzyme that improves resistance to proteolysis. These results establish conservation in both the catalytic mechanism and overall structure of Trm5 between evolutionarily distant eukaryotic and archaeal species and validate the crystal structure of the archaeal enzyme as a useful model for studies of the human enzyme.     

Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k_cat^app/K_M probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K_M and k_cat^app are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k_cat^app, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.     

Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases

Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC–MS/MS). Steady-state kinetics experiments showed similar k_cat and K_M values for both enzymes. In addition, similar IC₅₀ values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.     

Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternate Conformation of NADPH That May Be Linked to Trimethoprim Resistance

Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of β-hydroxyacyl-ACP dehydratase (FabZ)

Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The β-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence.