Aminoglycoside Nucleotidyltransferase Research Articles

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia.

Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 μM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Genes Vary Greatly in Their Propensity for Collateral Fitness Effects of Mutations.

Mutations can have deleterious fitness effects when they decrease protein specific activity or decrease active protein abundance. Mutations will also be deleterious when they cause misfolding or misinteractions that are toxic to the cell (i.e., independent of whether the mutations affect specific activity and abundance). The extent to which protein evolution is shaped by these and other collateral fitness effects is unclear in part because little is known of their frequency and magnitude. Using deep mutational scanning (DMS), we previously found at least 42% of missense mutations in the TEM-1 β-lactamase antibiotic resistance gene cause deleterious collateral fitness effects. Here, we used DMS to comprehensively determine the collateral fitness effects of missense mutations in three genes encoding the antibiotic resistance proteins New Delhi metallo-β-lactamase (NDM-1), chloramphenicol acetyltransferase I (CAT-I), and 2″-aminoglycoside nucleotidyltransferase (AadB). AadB (20%), CAT-I (0.9%), and NDM-1 (0.2%) were less susceptible to deleterious collateral fitness effects than TEM-1 (42%) indicating that genes have different propensities for these effects. As was observed with TEM-1, all the studied deleterious aadB mutants increased aggregation. However, aggregation did not correlate with collateral fitness effects for many of the deleterious mutants of CAT-I and NDM-1. Select deleterious mutants caused unexpected phenotypes to emerge. The introduction of internal start codons in CAT-1 caused loss of the episome and a mutation in aadB made its cognate antibiotic essential for growth. Our study illustrates how the complexity of the cell provides a rich environment for collateral fitness effects and new phenotypes to emerge.

Virulence gene profiles and antimicrobial susceptibility of Salmonella Brancaster from chicken.

The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [blaTEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

Whole-genome sequencing of Aminoglycoside nucleotidyltransferase aadA1 in patients with typhoid

S. typhi is causes typhoid in Iraq and developing countries. The abuse of antibiotics make the patient infected with S. typhi wich is resistant to many antibiotics. Aims of the study: Molecular and phylogenetic tree of genomic resistance genes associated Aminoglycoside nucleotidyltransferase (aad-A1), and detection mutation occur in aadA1 gene. Disc diffusion method and VITEK2 compact system to detect the resistance of S.typhi to 11 antibiotics and using the PCR technique to detect the prevalence of aadA1 resistance gene. The genomic DNA extracted from all S. typhi isolated harboring resistance gene. The prevalence of the aadA1 gene was 12(24%) from all S. typhi isolates and the presence of a silent mutation in sample number 2 Iraqi isolate. The first isolate (MW805237.1) was almost just like the isolates of each of the countries Korea, Iran Iran, India, and Madagascar, but The second Iraqi isolate (MW805238.1) was the share of congruence between them at 99% at position G. The aadA1 gene was present in S. typhi isolated from blood, the stool of human, and the detected mutation has no effect.

AadA36, a novel chromosomal aminoglycoside nucleotidyltransferase from a clinical isolate of Providencia stuartii.

In this study, we characterized a novel chromosome-encoded aminoglycoside nucleotidyltransferase (ANT), AadA36, from the Providencia stuartii strain P14 isolated from the sputum specimen of a burn patient at a hospital in Wenzhou, China. Among the functionally characterized ANTs, AadA36 shared the highest amino acid sequence identity of 51.91% with AadA14. The whole genome of P. stuartii P14 consisted of one chromosome and two plasmids (designated pP14-166 and pP14-114). A total of 19 genes with ≥80% similarity with functionally characterized antimicrobial resistance genes (ARGs) were identified in the whole genome, including aminoglycosides [aac(2')-Ia, aph(6)-Id, aph(3″)-Ib, aac(6')-Ib, ant(3″)-IIa, aph(3')-Ia], β-lactams (bla CMY-2 and bla OXA-10) and so on. Antimicrobial susceptibility testing showed that the aadA36 gene conferred specific resistance to spectinomycin and streptomycin, and the minimum inhibitory concentration (MIC) of these antimicrobials increased 128- and 64-fold compared with the control strain. The kinetic parameters of AadA36 were consistent with the MIC data of spectinomycin and streptomycin, with kcat /Km ratios of (1.07 ± 2.23) × 104 M-1 s-1 and (8.96 ± 1.01) × 103 M-1 s-1, respectively. The identification of a novel aminoglycoside resistance gene will help us further understand the complexity of the resistance mechanisms and provide deep insights into the dissemination of resistance genes in the microbial population.

Evaluation of The Antioxidant, Antimicrobial, and Anticancer Activities of Dicliptera bupleuroides Isolated Compounds Using In Vitro and In Silico Studies.

Phytochemical investigation of chloroform fraction (DBC) and ethyl acetate fraction (DBE) of D. bupleuroides (Acanthaceae) resulted in the isolation of β-sitosterol (1) from DBC and vanillic acid (2) from DBE, which were first to be isolated from D. bupleuroides. β-Sitosterol (1) exhibited substantial antioxidant activity (IC50 = 198.87 µg/mL), whereas vanillic acid (2) showed significant antioxidant power (IC50 = 92.68 µg/mL) employing 1,1-diphenyl-2-picrylhydrazyl (DPPH*) radical scavenging capacity assay. Both compounds showed pronounced antimicrobial activity using the agar disc diffusion method, particularly against fungi showing MIC values of 0.182 and 0.02 concerning Candida albicans, respectively, and 0.001 mg/mL regarding Penicillium notatum. They revealed considerable antibacterial activity with MIC values ranging between 0.467 and 0.809 mg/mL. Vanillic acid (2) exhibited substantial anticancer potential displaying 48.67% cell viability at a concentration of 100 μg/mL using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyl-2H-Tetrazolium Bromide) assay concerning HepG2 cell lines. These results were further consolidated by in silico studies on different enzymes, where vanillic acid displayed a high fitting score in the active pockets of DNA-gyrase, dihydrofolate reductase, aminoglycoside nucleotidyltransferase, and β-lactamase. It also inhibited human cyclin-dependent kinase 2 (CDK-2) and DNA topoisomerase II, as revealed by the in silico studies. ADME/TOPKAT (absorption, distribution, metabolism, excretion, and toxicity) prediction showed that vanillic acid exhibited reasonable pharmacodynamic, pharmacokinetic, and toxicity properties and, thus, could perfectly together with D. bupleuroides crude extract be incorporated in pharmaceutical preparations to counteract cancer and microbial invasion, as well as oxidative stress. Thus, it is concluded that D. bupleroides could be a potential source of therapeutically active compounds, which would be helpful for the discovery of clinically effective and safe drugs.

Bioactive Alkaloids from Genus Aspergillus: Mechanistic Interpretation of Their Antimicrobial and Potential SARS-CoV-2 Inhibitory Activity Using Molecular Modelling.

Genus Aspergillus represents a widely spread genus of fungi that is highly popular for possessing potent medicinal potential comprising mainly antimicrobial, cytotoxic and antioxidant properties. They are highly attributed to its richness by alkaloids, terpenes, steroids and polyketons. This review aimed to comprehensively explore the diverse alkaloids isolated and identified from different species of genus Aspergillus that were found to be associated with different marine organisms regarding their chemistry and biology. Around 174 alkaloid metabolites were reported, 66 of which showed important biological activities with respect to the tested biological activities mainly comprising antiviral, antibacterial, antifungal, cytotoxic, antioxidant and antifouling activities. Besides, in silico studies on different microbial proteins comprising DNA-gyrase, topoisomerase IV, dihydrofolate reductase, transcriptional regulator TcaR (protein), and aminoglycoside nucleotidyl transferase were done for sixteen alkaloids that showed anti-infective potential for better mechanistic interpretation of their probable mode of action. The inhibitory potential of compounds vs. Angiotensin-Converting Enzyme 2 (ACE2) as an important therapeutic target combating COVID-19 infection and its complication was also examined using molecular docking. Fumigatoside E showed the best fitting within the active sites of all the examined proteins. Thus, Aspergillus species isolated from marine organisms could afford bioactive entities combating infectious diseases.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases

ABSTRACT The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase – (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5ʹ UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5ʹ UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

Structural Recognition of Spectinomycin by Resistance Enzyme ANT(9) from Enterococcus faecalis.

Spectinomycin is a ribosome-binding antibiotic that blocks the translocation step of translation. A prevalent resistance mechanism is modification of the drug by aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific ANT(9) family or by enzymes of the dual-specificity ANT(3")(9) family, which also acts on streptomycin. We previously reported the structural mechanism of streptomycin modification by the ANT(3")(9) AadA from Salmonella enterica ANT(9) from Enterococcus faecalis adenylates the 9-hydroxyl of spectinomycin. Here, we present the first structures of spectinomycin bound to an ANT enzyme. Structures were solved for ANT(9) in apo form, in complex with ATP, spectinomycin, and magnesium, or in complex with only spectinomycin. ANT(9) shows an overall structure similar to that of AadA, with an N-terminal nucleotidyltransferase domain and a C-terminal α-helical domain. Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is buried at the bottom. Upon drug binding, the C-terminal domain rotates 14 degrees to close the cleft, allowing contacts of both domains with the drug. Comparison with AadA shows that spectinomycin specificity is explained by a straight α5 helix and a shorter α5-α6 loop, which would clash with the larger streptomycin substrate. In the active site, we observed two magnesium ions, one of them in a previously unobserved position that may activate the 9-hydroxyl for deprotonation by the catalytic base Glu-86. The observed binding mode for spectinomycin suggests that spectinamides and aminomethyl spectinomycins, recent spectinomycin analogues with expansions in position 4 of the C ring, are also subjected to modification by ANT(9) and ANT(3")(9) enzymes.

"Catch and Release": A Variation of the Archetypal Nucleotidyl Transfer Reaction.

Nucleotidyl transfer is an archetypal enzyme reaction central to DNA replication and repair. Here we describe a variation of the nucleotidylation reaction termed "catch and release" that is used by an antibiotic modifying enzyme. The aminoglycoside nucleotidyl transferase 4' (ANT4') inactivates antibiotics such as kanamycin and neomycin through nucleotidylation within an active site that shares significant structural, and inferred underlying catalytic similarity, with human DNA polymerase beta. Here we follow the entire nucleotidyl transfer reaction coordinate of ANT4' covalently inactivating neomycin using X-ray crystallography. These studies show that although the underlying reaction mechanism is conserved with polymerases, a short 2.35 A hydrogen bond is initially formed to facilitate tight binding of the aminoglycoside substrate and is subsequently disrupted by the assembly of the catalytically active ternary complex. This enables the release of products post catalysis due to a lower free energy of the product state compared to the starting substrate complex. We propose that this "catch and release" mechanism of antibiotic turnover observed in ANT4' is a variation of nucleotidyl transfer that has been adapted for the inactivation of antibiotics.

Structural characterization of aminoglycoside 4'-O-adenylyltransferase ANT(4')-IIb from Pseudomonas aeruginosa.

Aminoglycosides were one of the first classes of broad-spectrum antibacterial drugs clinically used to effectively combat infections. The rise of resistance to these drugs, mediated by enzymatic modification, has since compromised their utility as a treatment option, prompting intensive research into the molecular function of resistance enzymes. Here, we report the crystal structure of aminoglycoside nucleotidyltransferase ANT(4')-IIb in apo and tobramycin-bound forms at a resolution of 1.6 and 2.15 Å, respectively. ANT(4')-IIb was discovered in the opportunistic pathogen Pseudomonas aeruginosa and conferred resistance to amikacin and tobramycin. Analysis of the ANT(4')-IIb structures revealed a two-domain organization featuring a mixed β-sheet and an α-helical bundle. ANT(4')-IIb monomers form a dimer required for its enzymatic activity, as coordination of the aminoglycoside substrate relies on residues contributed by both monomers. Despite harbouring appreciable primary sequence diversity compared to previously characterized homologues, the ANT(4')-IIb structure demonstrates a surprising level of structural conservation highlighting the high plasticity of this general protein fold. Site-directed mutagenesis of active site residues and kinetic analysis provides support for a catalytic mechanism similar to those of other nucleotidyltransferases. Using the molecular insights provided into this ANT(4')-IIb-represented enzymatic group, we provide a hypothesis for the potential evolutionary origin of these aminoglycoside resistance determinants.

Evolutionary significance and functional characterization of streptomycin adenylyltransferase from Serratia marcescens

Complete functional annotations of proteins are essential to understand the role and mechanisms in pathogenesis. Aminoglycoside nucleotidyltransferases are the subclasses of aminoglycosides modifying enzymes conferring resistance to organisms. Insight into the structural and functional understanding of nucleotidyltransferase family protein provides vital information to combat pathogenesis. Phylogenetic analysis is employed to identify the evolutionary significance and common motif’s present among the homologs of nucleotidyltransferase family protein. Structure, sequence based approaches and molecular docking were implemented to predict the exact function of the protein. Wide distribution of the nucleotidyltransferase family protein in gram-positive and gram-negative organisms are evidenced from phylogenetic analysis. Five common motifs were present in all the homolog’s of nucleotidyltransferase family protein. Sequence-structure based functional annotations predicts that the targeted protein function as ATP-Mg dependent streptomycin adenylyltransferase. Structural comparisons and docking studies correlate well with the identified function. The complete function of nucleotidyltransferase family protein was identified as Streptomycin adenylyltransferase and it could be targeted as a potential therapeutic target to overcome antibiotic resistance.Communicated by Ramaswamy H. SarmaAbbreviationsAACaminoglycoside acetyltransferasesAMEaminoglycoside modifying enzymeANTaminoglycoside nucleotidyltransferasesAPHaminoglycoside phosphotransferasesATPadenosine triphosphateCASTpcomputer atlas and surface topography of proteinsDUFdomains of unknown functionGlidegrid-based ligand docking with energeticHMMhidden Markov modelMASTmotif alignment and search toolMEGAmolecular evolutionary genetics analysisMEMEmultiple Em for motif elicitationMSAmultiple sequence alignmentNMPnucleoside monophosphateNTPnucleoside triphosphateNTnucleotidyltransferaseOPLSoptimized potential for liquid simulationXPextra precision

Characterization of the Microbial Resistome in Conventional and "Raised Without Antibiotics" Beef and Dairy Production Systems.

Metagenomic investigations have the potential to provide unprecedented insights into microbial ecologies, such as those relating to antimicrobial resistance (AMR). We characterized the microbial resistome in livestock operations raising cattle conventionally (CONV) or without antibiotic exposures (RWA) using shotgun metagenomics. Samples of feces, wastewater from catchment basins, and soil where wastewater was applied were collected from CONV and RWA feedlot and dairy farms. After DNA extraction and sequencing, shotgun metagenomic reads were aligned to reference databases for identification of bacteria (Kraken) and antibiotic resistance genes (ARGs) accessions (MEGARes). Differences in microbial resistomes were found across farms with different production practices (CONV vs. RWA), types of cattle (beef vs. dairy), and types of sample (feces vs. wastewater vs. soil). Feces had the greatest number of ARGs per sample (mean = 118 and 79 in CONV and RWA, respectively), with tetracycline efflux pumps, macrolide phosphotransferases, and aminoglycoside nucleotidyltransferases mechanisms of resistance more abundant in CONV than in RWA feces. Tetracycline and macrolide–lincosamide–streptogramin classes of resistance were more abundant in feedlot cattle than in dairy cow feces, whereas the β-lactam class was more abundant in dairy cow feces. Lack of congruence between ARGs and microbial communities (procrustes analysis) suggested that other factors (e.g., location of farms, cattle source, management practices, diet, horizontal ARGs transfer, and co-selection of resistance), in addition to antimicrobial use, could have impacted resistome profiles. For that reason, we could not establish a cause–effect relationship between antimicrobial use and AMR, although ARGs in feces and effluents were associated with drug classes used to treat animals according to farms’ records (tetracyclines and macrolides in feedlots, β-lactams in dairies), whereas ARGs in soil were dominated by multidrug resistance. Characterization of the “resistance potential” of animal-derived and environmental samples is the first step toward incorporating metagenomic approaches into AMR surveillance in agricultural systems. Further research is needed to assess the public-health risk associated with different microbial resistomes.

The Thermodynamics of Ligand Binding to the Aminoglycoside O-Nucleotidyltransferase(4') and Variants Yields Clues about Thermophilic Properties.

The aminoglycoside nucleotidyltransferase(4') is an enzyme with high substrate promiscuity and catalyzes the transfer of the AMP group from ATP to the 4'-OH site of many structurally diverse aminoglycosides, which results in the elimination of their effectiveness as antibiotics. Two thermostable variants carrying single-site mutations are used to determine the molecular properties associated with thermophilicity. The thermodynamics of enzyme-ligand interactions showed that one variant (T130K) has properties identical to those of the mesophilic wild type (WT) while the other (D80Y) behaved differently. Differences between D80Y and the T130K/WT pair include the change in heat capacity (Δ Cp), which is dependent on temperature for D80Y but not for WT or T130K. The change in Δ Cp with temperature (ΔΔ Cp) with D80Y is dependent on aminoglycoside only in H2O and remains the same with all aminoglycosides in D2O. Furthermore, the offset temperature ( Toff), the temperature difference that yields identical enthalpies in H2O and D2O, becomes larger with an increase in temperature for WT and T130K but remains mostly unchanged for D80Y. Studies in H2O and D2O revealed that solvent reorganization becomes the major contributor to ligand binding with an increase in temperature for WT and T130K, but changes in low-frequency vibrational modes are the main contributors with D80Y. Data presented in this paper suggest that global properties associated with the enzyme-ligand interactions, such as the thermodynamics of ligand binding, may yield clues about thermophilicity and permit us to distinguish those variants that are simply a more thermostable version of the mesophilic protein.

Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica.

Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3″)(9) adenylyltransferase that O-adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

Investigation of the Molecular Mechanisms which Result in Aminoglycoside Nucleotidyltransferase 4′ (ANT4) Variants with Different Levels of Thermostability

The aminoglycoside nucleotidyltransferase 4′ (ANT) is a homodimeric enzyme that detoxifies a large number of aminoglycoside antibiotics by nucleotidylating them at the C4′-OH site. Two thermostable variants for this enzyme show only single amino acid changes in their primary amino acid sequences (T130K, D80Y). D80Y has a higher melting temperature compared to T130K. Our previous studies indicate that T130K mimics the WT enzyme in terms of protein dynamics and thermodynamics of enzyme-ligand interactions while the D80Y exhibits different behaviors in both aspects. This led us to hypothesize that the T130K is just a thermostable version of the mesophilic WT exhibiting the same behaviors while the D80Y is the true thermophilic variant with distinct properties. Our recent work shows that the thermodynamic parameters of the binary enzyme-aminoglycoside complexes of these variants show highly significant differences. The data, acquired in H2O and D2O by isothermal titration calorimetry demonstrated that solvent reorganization upon ligand binding show large differences between the two variants. The heat capacity change (ΔCp) also show antibiotic-dependent differences between the two variants. The X-ray structures of both variants show that the aminoglycoside neomycin is positioned differently at the active site of T130K as compared to D80Y, in which the tyrosine substitution alters the geometry of binding site. This structural difference explains why we observe differences in the thermodynamics of the ligand binding properties between these variants. Furthermore, temperature affects the monomer-dimer equilibrium of these enzymes differently. Thus, data shown in this work suggest that dynamic properties of proteins, thermodynamics of ligand-protein interactions, solvent effects and monomer-dimer equilibrium may be among the molecular parameters that separate thermophilic proteins from simply those that are thermostable but otherwise identical to the mesophilic counterparts.

Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line?

Aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that modifies the C4'-OH site of aminoglycoside antibiotics by nucleotidylation. A few single- and double-residue mutants of this enzyme (T130K, D80Y, and D80Y/T130K) from Bacillus stearothermophilus show increased thermostability. This article investigates how such residue replacements, which are distant from the active site and monomer-monomer interface, result in various changes of the thermostability of the enzyme. In this work, we show that the thermodynamic properties of enzyme-ligand complexes and protein dynamics may be indicators of a thermophilic behavior. Our data suggests that one of the single-site mutants of ANT, D80Y, may be a thermophilic protein and the other thermostable mutant, T130K, is actually a more heat-stable variant of the mesophilic wild type (WT) with a higher Tm. Our data also suggest that T130K and D80Y adopt different global dynamics strategies to achieve different levels of thermostability enhancement and that the differences between the properties of the species can be described in terms of global dynamics rather than in terms of specific structural features. Thermophilicity of the D80Y comes at the cost of less favorable thermodynamic parameters for ligand binding relative to WT. On the other hand, the T130K species exhibits the same affinity to ligands and the same thermodynamic parameters of complex formation as the WT enzyme. These observations suggest that a quantitative characterization of ligand binding and protein dynamics can be used to differentiate thermophilic proteins from their simply more heat-stable mesophilic counterparts.

Occurrence and clonal diversity of multidrug-resistant Klebsiella pneumoniae recovered from inanimate surfaces in Algerian hospital environment: First report of armA, qnrB and aac(6')-Ib-cr genes.

The aim of this study is to characterize the molecular support of antibiotic resistance in MDR Klebsiella pneumoniae recovered from inanimate surfaces between March 2012 to February 2014 in three teaching hospitals (Setif, Bejaia and Constantine) in Algeria. Forty-four K. pneumoniae producing ESBL were detected and blaCTX-M-15 and blaCTX-M-3 were detected respectively in 41 and 3 isolates. These K. pneumoniae isolates producing ESBL were also resistant to gentamicin (87%), tobramicin (87%), ciprofloxacin (66%) and ofloxacin (62%). Aminoglycosides resistance genes detected were 16S rRNA methylase (armA), aminoglycoside acetyl-transferase (aac(6')-Ib), aminoglycoside nucleotidyl-transferase (aadA2) and aminoglycoside, phosphoryl-transferase (ant3″Ih-aac(6')-IId). Plasmid-mediated quinolone resistance (PMQR) genes detected were aac(6')-Ib-cr (34 isolates) and qnrB genes in (34 isolates). Multilocus sequence typing (MLST) resulted in 12 different sequence types (STs) regrouped into 5 clonal complexes (CC147, CC17, CC37, CC2 and CC23), one clonal group (CG485) and 4 singletons (ST1426, ST405, ST1308, ST873). Here, we report the detection of the ESBLs encoding gene linked with plasmid-mediated quinolone resistance (PMQR) and aminoglycosides resistance recovered from inanimate surfaces in hospital environment.

A new subclass of intrinsic aminoglycoside nucleotidyltransferases, ANT(3")-II, is horizontally transferred among Acinetobacter spp. by homologous recombination.

The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes.

The Role of Solvent Effect on the Ligand Binding Properties of the Thermostable Variants of Aminoglycoside Nucleotidyltransferase 4¯(ANT4)

The Role of Solvent Effect on the Ligand Binding Properties of the Thermostable Variants of Aminoglycoside Nucleotidyltransferase 4¯(ANT4)