Structure and Mechanism of the Lincosamide Antibiotic Adenylyltransferase LinB

SummaryLincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate– and binary product–bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve

Crystal structure of an aminoglycoside 6′-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold

AbstractBackground: The predominant mechanism of antibiotic resistance employed by pathogenic bacteria against the clinically used aminoglycosides is chemical modification of the drug. The detoxification reactions are catalyzed by enzymes that promote either the phosphorylation, adenylation or acetylation of aminoglycosides. Structural studies of these aminoglycoside-modifying enzymes may assist in the development of therapeutic agents that could circumvent antibiotic resistance. In addition, such studies may shed light on the development of antibiotic resistance and the evolution of different enzyme classes.Results: The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside 6′-N-acetyltransferase type Ii (AAC(6′)-Ii) in complex with the cofactor acetyl coenzyme A has been determined at 2.7 Å resolution. The structure establishes that this acetyltransferase belongs to the GCN5-related N-acetyltransferase superfamily, which includes such enzymes as the histone acetyltransferases GCN5 and Hat1.Conclusions: Comparison of the AAC(6′)-Ii structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6′)-Ii possesses protein acetylation activity. Thus, AAC(6′)-Ii is both a structural and functional homolog of the GCN5-related histone acetyltransferases

A Cryptic Polyene Biosynthetic Gene Cluster in Streptomyces calvus Is Expressed upon Complementation with a Functional bldA Gene

SummaryStreptomyces calvus is best known as the producer of the fluorinated natural product nucleocidin. This strain of Streptomycetes is also unusual for displaying a “bald” phenotype that is deficient in the formation of aerial mycelium and spores. Genome sequencing of this organism revealed a point mutation in the bldA gene that is predicted to encode a misfolded Leu-tRNAUUA molecule. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a polyeneoic acid amide, 4-Z-annimycin, and a minor amount of the isomer, 4-E-annimycin. Bioassays reveal that these compounds inhibit morphological differentiation in other Actinobacteria. The annimycin gene cluster encoding a type 1 polyketide synthase was identified and verified through disruption studies. This study underscores the importance of the bldA gene in regulating the expression of cryptic biosynthetic genes

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment

Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole

The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies

Nova-like Cataclysmic Variables in the Infrared

Novalike cataclysmic variables have persistently high mass transfer rates and prominent steady state accretion disks. We present an analysis of infrared observations of twelve novalikes obtained from the Two Micron All Sky Survey, the Spitzer Space Telescope, and the Wide-field Infrared Survey Explorer All Sky Survey. The presence of an infrared excess at >3-5 microns over the expectation of a theoretical steady state accretion disk is ubiquitous in our sample. The strength of the infrared excess is not correlated with orbital period, but shows a statistically significant correlation (but shallow trend) with system inclination that might be partially (but not completely) linked to the increasing view of the cooler outer accretion disk and disk rim at higher inclinations. We discuss the possible origin of the infrared excess in terms of emission from bremsstrahlung or circumbinary dust, with either mechanism facilitated by the mass outflows (e.g., disk wind/corona, accretion stream overflow, and so on) present in novalikes. Our comparison of the relative advantages and disadvantages of either mechanism for explaining the observations suggests that the situation is rather ambiguous, largely circumstantial, and in need of stricter observational constraints.Peer reviewe

When Antibiotics Fail: The Expert Panel on the Potential Socio-Economic Impacts of Antimicrobial Resistance in Canada

Antimicrobials are life savers in Canada, enabling modern healthcare and playing a central role in agriculture. They have reduced the economic, medical, and social burden of infectious diseases and are part of many routine medical interventions, such as caesarean sections, joint replacements, and tonsillectomies. As use of antimicrobials has increased, bacteria evolved to become resistant, resulting in drugs that are no longer effective at treating infections. Antimicrobial resistance (AMR) is increasing worldwide, and with widespread trade and travel, resistance can spread quickly, posing a serious threat to all countries. For Canada, the implications of AMR are stark. When Antibiotics Fail examines the current impacts of AMR on our healthcare system, projects the future impact on Canada’s GDP, and looks at how widespread resistance will influence the day-to-day lives of Canadians. The report examines these issues through a One Health lens, recognizing the interconnected nature of AMR, from healthcare settings to the environment to the agriculture sector. It is the most comprehensive report to date on the economic impact of AMR in Canada