Decarboxylation of the Catalytic Lysine Residue by the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the OXA-58 Carbapenemase.

Antibiotic resistance in bacteria is a major global health concern. The wide spread of carbapenemases, bacterial enzymes that degrade the last-resort carbapenem antibiotics, is responsible for multidrug resistance in bacterial pathogens and has further significantly exacerbated this problem. is one of the leading nosocomial pathogens due to the acquisition and wide dissemination of carbapenem-hydrolyzing class D β-lactamases, which have dramatically diminished available therapeutic options.

Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase.

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed.

The C5α-Methyl-Substituted Carbapenem NA-1-157 Exhibits Potent Activity against spp. Isolates Producing OXA-48-Type Carbapenemases.

The wide spread of carbapenem-hydrolyzing β-lactamases in Gram-negative bacteria has diminished the utility of the last-resort carbapenem antibiotics, significantly narrowing the available therapeutic options. In the family, which includes many important clinical pathogens such as and , production of class D β-lactamases from the OXA-48-type family constitutes the major mechanism of resistance to carbapenems. To address the public health threat posed by these enzymes, novel, effective therapeutics are urgently needed.

The l,d-Transpeptidase Ldt from Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of Ldt from the clinically important pathogen, .

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Carbapenemase OXA-23 by Impeding Deacylation.

Acinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to β-lactam antibiotics by producing β-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of β-lactam antibiotics have been the drugs of choice for treating A.

Effects of Inactivation of d,d-Transpeptidases of Acinetobacter baumannii on Bacterial Growth and Susceptibility to β-Lactam Antibiotics.

Resistance to β-lactams, the most used antibiotics worldwide, constitutes the major problem for the treatment of bacterial infections. In the nosocomial pathogen Acinetobacter baumannii, β-lactamase-mediated resistance to the carbapenem family of β-lactam antibiotics has resulted in the selection and dissemination of multidrug-resistant isolates, which often cause infections characterized by high mortality rates. There is thus an urgent demand for new β-lactamase-resistant antibiotics that also inhibit their targets, penicillin-binding proteins (PBPs).

Time-Resolved Interaction of the CDD-1 enzyme with Avibactam Provides New Insights into the Catalytic Mechanism of Class D β-lactamases.

Class D β-lactamases have risen to notoriety due to their wide spread in bacterial pathogens, propensity to inactivate clinically important β-lactam antibiotics, and ability to withstand inhibition by the majority of classical β-lactamase inhibitors. Understanding the catalytic mechanism of these enzymes is thus vitally important for the development of novel antibiotics and inhibitors active against infections caused by antibiotic-resistant bacteria. Here we report an time-resolved study of the interaction of the class D β-lactamase CDD-1 from with the diazobicyclooctane inhibitor, avibactam.

Inhibition of the Class D β-Lactamase CDD-1 by Avibactam.

Avibactam is a potent diazobicyclooctane inhibitor of class A and C β-lactamases. The inhibitor also exhibits variable activity against some class D enzymes from Gram-negative bacteria; however, its interaction with recently discovered class D β-lactamases from Gram-positive bacteria has not been studied. Here, we describe microbiological, kinetic, and mass spectrometry studies of the interaction of avibactam with CDD-1, a class D β-lactamase from the clinically important pathogen , and show that avibactam is a potent irreversible mechanism-based inhibitor of the enzyme.

A surface loop modulates activity of the Bacillus class D β-lactamases.

The expression of β-lactamases is a major mechanism of bacterial resistance to the β-lactam antibiotics. Four molecular classes of β-lactamases have been described (A, B, C and D), however until recently the class D enzymes were thought to exist only in Gram-negative bacteria. In the last few years, class D enzymes have been discovered in several species of Gram-positive microorganisms, such as Bacillus and Clostridia, and an investigation of their kinetic and structural properties has begun in earnest.

Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases.

Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor.

The crystal structures of CDD-1, the intrinsic class D β-lactamase from the pathogenic Gram-positive bacterium Clostridioides difficile, and its complex with cefotaxime.

Class D β-lactamases, enzymes that degrade β-lactam antibiotics and are widely spread in Gram-negative bacteria, were for a long time not known in Gram-positive organisms. Recently, these enzymes were identified in various non-pathogenic Bacillus species and subsequently in Clostridioides difficile, a major clinical pathogen associated with high morbidity and mortality rates. Comparison of the BPU-1 enzyme from Bacillus pumilus with the CDD-1 and CDD-2 enzymes from C.

Structural Insights into the Mechanism of Carbapenemase Activity of the OXA-48 β-Lactamase.

Carbapenem-hydrolyzing class D carbapenemases (CHDLs) are enzymes that produce resistance to the last-resort carbapenem antibiotics, severely compromising the available therapeutic options for the treatment of life-threatening infections. A broad variety of CHDLs, including OXA-23, OXA-24/40, and OXA-58, circulate in , while the OXA-48 CHDL is predominant in Extensive structural studies of enzymes have provided important information regarding their interactions with carbapenems and significantly contributed to the understanding of the mechanism of their carbapenemase activity. However, the interactions between carbapenems and OXA-48 have not yet been elucidated.

Intrinsic Class D β-Lactamases of .

is the causative agent of the deadly infection. Resistance of the pathogen to β-lactam antibiotics plays a major role in the development of the disease, but the mechanism of resistance is currently unknown. We discovered that encodes class D β-lactamases, i.

Role of the Hydrophobic Bridge in the Carbapenemase Activity of Class D β-Lactamases.

Class D carbapenemases are enzymes of the utmost clinical importance due to their ability to confer resistance to the last-resort carbapenem antibiotics. We investigated the role of the conserved hydrophobic bridge in the carbapenemase activity of OXA-23, the major carbapenemase of the important pathogen We show that substitution of the bridge residue Phe110 affects resistance to meropenem and doripenem and has little effect on MICs of imipenem. The opposite effect was observed upon substitution of the other bridge residue Met221.

Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im.

Aminoglycoside 6'-acetyltransferase-Im (AAC(6')-Im) is the closest monofunctional homolog of the AAC(6')-Ie acetyltransferase of the bifunctional enzyme AAC(6')-Ie/APH(2")-Ia. The AAC(6')-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6')-Ie, yet unlike AAC(6')-Ie, the AAC(6')-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6')-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6' amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor.

The role of conserved surface hydrophobic residues in the carbapenemase activity of the class D β-lactamases.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) produce resistance to the last-resort carbapenem antibiotics and render these drugs ineffective for the treatment of life-threatening infections. Here, it is shown that among the clinically important CHDLs, OXA-143 produces the highest levels of resistance to carbapenems and has the highest catalytic efficiency against these substrates. Structural data demonstrate that acylated carbapenems entirely fill the active site of CHDLs, leaving no space for water molecules, including the deacylating water.

Role of the Conserved Disulfide Bridge in Class A Carbapenemases.

Some members of the class A β-lactamase family are capable of conferring resistance to the last resort antibiotics, carbapenems. A unique structural feature of these clinically important enzymes, collectively referred to as class A carbapenemases, is a disulfide bridge between invariant Cys and Cys residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated.

Class D β-lactamases do exist in Gram-positive bacteria.

Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms.

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D β-Lactamases.

Class D β-lactamases of Acinetobacter baumannii are enzymes of the utmost clinical importance, producing resistance to last resort carbapenem antibiotics. Although the OXA-51-like enzymes constitute the largest family of class D β-lactamases, they are poorly studied and their importance in conferring carbapenem resistance is controversial. We present the detailed microbiological, kinetic, and structural characterization of the eponymous OXA-51 β-lactamase.

Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases.

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics.

Class D β-lactamases: are they all carbapenemases?

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified in Acinetobacter spp., notably in Acinetobacter baumannii.

Structural basis for the MukB-topoisomerase IV interaction and its functional implications in vivo.

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB-topo IV complex to 2.3 Å resolution.

Structural basis for carbapenemase activity of the OXA-23 β-lactamase from Acinetobacter baumannii.

Dissemination of Acinetobacter baumannii strains harboring class D β-lactamases producing resistance to carbapenem antibiotics severely limits our ability to treat deadly Acinetobacter infections. Susceptibility determination in the A. baumannii background and kinetic studies with a homogeneous preparation of OXA-23 β-lactamase, the major carbapenemase present in A.

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction.

In contrast to the current state of knowledge in the field of eukaryotic chromosome segregation, relatively little is known about the mechanisms coordinating the appropriate segregation of bacterial chromosomes. In Escherichia coli, the MukB/E/F complex and topoisomerase IV (Topo IV) are both crucial players in this process. Topo IV removes DNA entanglements following the replication of the chromosome, whereas MukB, a member of the structural maintenance of chromosomes protein family, serves as a bacterial condensin.