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

Resistance to ß-lactams, the most used antibiotics worldwide, constitutes the major problem for the treatment of bacterial infections. In the nosocomial pathogen Acinetobacter baumannii, ß-lactamase-mediated resistance to the carbapenem family of ß-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 ß-lactamase-resistant antibiotics that also inhibit their targets, penicillin-binding proteins (PBPs). As some PBPs are indispensable for the biosynthesis of the bacterial cell wall and survival, we evaluated their importance for the growth of A. baumannii by performing gene inactivation studies of d,d-transpeptidase domains of high-molecular-mass (HMM) PBPs individually and in combination with one another. We show that PBP3 is essential for A. baumannii survival, as deletion mutants of this d,d-transpeptidase were not viable. The inactivation of PBP1a resulted in partial cell lysis and retardation of bacterial growth, and these effects were further enhanced by the additional inactivation of PBP2 but not PBP1b. Susceptibility to ß-lactam antibiotics increased 4- to 8-fold for the A. baumannii PBP1a/PBP1b/PBP2 triple mutant and 2- to 4-fold for all remaining mutants. Analysis of the peptidoglycan structure revealed a significant change in the muropeptide composition of the triple mutant and demonstrated that the lack of d,d-transpeptidase activity of PBP1a, PBP1b, and PBP2 is compensated for by an increase in the l,d-transpeptidase-mediated cross-linking activity of LdtJ. Overall, our data showed that in addition to essential PBP3, the simultaneous inhibition of PBP1a and PBP2 or PBPs in combination with LdtJ could represent potential strategies for the design of novel drugs against A. baumannii.

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

The expression of ß-lactamases is a major mechanism of bacterial resistance to the ß-lactam antibiotics. Four molecular classes of ß-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. Interestingly, it was observed that some species of Bacillus produce two distinct class D ß-lactamases, one highly active and the other with only basal catalytic activity. Analysis of amino acid sequences of active (BPU-1 from Bacillus pumilus) and inactive (BSU-2 from Bacillus subtilis and BAT-2 from Bacillus atrophaeus) enzymes suggests that presence of three additional amino acid residues in one of the surface loops of inefficient ß-lactamases may be responsible for their severely diminished activity. Our structural and docking studies show that the elongated loop of these enzymes severely restricts binding of substrates. Deletion of the three residues from the loops of BSU-2 and BAT-2 ß-lactamases relieves the steric hindrance and results in a significant increase in the catalytic activity of the enzymes. These data show that this surface loop plays an important role in modulation of the catalytic activity of Bacillus class D ß-lactamases.

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

Class D ß-lactamases, enzymes that degrade ß-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. difficile demonstrated that the latter enzymes have broadened their substrate profile to efficiently hydrolyze the expanded-spectrum methoxyimino cephalosporins, cefotaxime and ceftriaxone. These two antibiotics are major contributors to the development of C. difficile infection, as they suppress sensitive bacterial microflora in the gut but fail to kill the pathogen which is highly resistant to these drugs. To gain insight into the structural features that contribute to the expansion of the substrate profile of CDD enzymes compared to BPU-1, we solved the crystal structures of CDD-1 and its complex with cefotaxime. Comparison of CDD-1 structures with those of class D enzymes from Gram-negative bacteria showed that in the cefotaxime-CDD-1 complex, the antibiotic is bound in a substantially different mode due to structural differences in the enzymes' active sites. We also found that CDD-1 has a uniquely long Ω-loop when compared to all other class D ß-lactamases. This Ω-loop extension allows it to engage in hydrogen bonding with the acylated cefotaxime, thus providing additional stabilizing interactions with the substrate which could be responsible for the high catalytic activity of the enzyme for expanded-spectrum cephalosporins.

Structural Insights into the Mechanism of Carbapenemase Activity of the OXA-48 ß-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 Acinetobacter baumannii, while the OXA-48 CHDL is predominant in Enterobacteriaceae Extensive structural studies of A. baumannii 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. We determined the X-ray crystal structures of the acyl-enzyme complexes of OXA-48 with four carbapenems, imipenem, meropenem, ertapenem, and doripenem, and compared them with those of known carbapenem complexes of A. baumannii CHDLs. In the A. baumannii enzymes, acylation by carbapenems triggers significant displacement of one of two conserved hydrophobic surface residues, resulting in the formation of a channel for entry of the deacylating water into the active site. We show that such a channel preexists in apo-OXA-48 and that only minor displacement of the conserved hydrophobic surface residues occurs upon the formation of OXA-48 acyl-enzyme intermediates. We also demonstrate that the extensive hydrophobic interactions that occur between a conserved hydrophobic bridge of the A. baumannii CHDLs and the carbapenem tails are lost in OXA-48 in the absence of an equivalent bridge structure. These data highlight significant differences between the interactions of carbapenems with OXA-48 and those with A. baumannii enzymes and provide important insights into the mechanism of carbapenemase activity of the major Enterobacteriaceae CHDL, OXA-48.

Role of the Hydrophobic Bridge in the Carbapenemase Activity of Class D ß-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 Acinetobacter baumannii 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. Complete disruption of the bridge by the F110A/M221A substitution resulted in a significant loss of affinity for doripenem and meropenem and to a lesser extent for imipenem, which is reflected in the reduced MICs of these antibiotics. In the wild-type OXA-23, the pyrrolidine ring of the meropenem tail forms a hydrophobic interaction with Phe110 of the bridge. Similar interactions would ensue with ring-containing doripenem but not with imipenem, which lacks this ring. Our structural studies showed that this interaction with the meropenem tail is missing in the F110A/M221A mutant. These data explain why disruption of the interaction between the enzyme and the carbapenem substrate impacts the affinity and MICs of meropenem and doripenem to a larger degree than those of imipenem. Our structures also show that the bridge directs the acylated carbapenem into a specific tautomeric conformation. However, it is not this conformation but rather the stabilizing interaction between the tail of the antibiotic and the hydrophobic bridge that contributes to the carbapenemase activity of class D ß-lactamases.

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

Production of ß-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to ß-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 ß-lactamases capable of hydrolyzing a wide array of ß-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D ß-lactamases. These enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.

Role of the Conserved Disulfide Bridge in Class A Carbapenemases.

Some members of the class A ß-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 Cys69 and Cys238 residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated. Here we show that disruption of the disulfide bridge in the GES-5 carbapenemase by the C69G substitution results in only minor decreases in the conferred levels of resistance to the carbapenem imipenem and other ß-lactams. Kinetic and circular dichroism experiments with C69G-GES-5 demonstrate that this small drop in antibiotic resistance is due to a decline in the enzyme activity caused by a marginal loss of its thermal stability. The atomic resolution crystal structure of C69G-GES-5 shows that two domains of this disulfide bridge-deficient enzyme are held together by an intensive hydrogen-bonding network. As a result, the protein architecture and imipenem binding mode remain unchanged. In contrast, the corresponding hydrogen-bonding networks in NMCA, SFC-1, and SME-1 carbapenemases are less intensive, and as a consequence, disruption of the disulfide bridge in these enzymes destabilizes them, which causes arrest of bacterial growth. Our results demonstrate that the disulfide bridge is essential for stability but does not play a direct role in the carbapenemase activity of the GES family of ß-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity.

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

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES ß-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. This highlights the increasing need to understand the mechanisms by which the GES ß-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to an increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem-GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 ß-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES ß-lactamases. Our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.

Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia revealed by crystallographic and small-angle X-ray scattering analysis.

Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6')-Ie-APH(2'')-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6')-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2'')-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2'')-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6')-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6')-Ie enzyme is joined to APH(2'')-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2'')-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6')-Ie-APH(2'')-Ia enzyme.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Šresolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

Structure of the extended-spectrum class C ß-lactamase ADC-1 from Acinetobacter baumannii.

ADC-type class C ß-lactamases comprise a large group of enzymes that are encoded by genes located on the chromosome of Acinetobacter baumannii, a causative agent of serious bacterial infections. Overexpression of these enzymes renders A. baumannii resistant to various ß-lactam antibiotics and thus severely compromises the ability to treat infections caused by this deadly pathogen. Here, the high-resolution crystal structure of ADC-1, the first member of this clinically important family of antibiotic-resistant enzymes, is reported. Unlike the narrow-spectrum class C ß-lactamases, ADC-1 is capable of producing resistance to the expanded-spectrum cephalosporins, rendering them inactive against A. baumannii. The extension of the substrate profile of the enzyme is likely to be the result of structural differences in the R2-loop, primarily the deletion of three residues and subsequent rearrangement of the A10a and A10b helices. These structural rearrangements result in the enlargement of the R2 pocket of ADC-1, allowing it to accommodate the bulky R2 substituents of the third-generation cephalosporins, thus enhancing the catalytic efficiency of the enzyme against these clinically important antibiotics.

Crystal structure of carbapenemase OXA-58 from Acinetobacter baumannii.

Class D ß-lactamases capable of hydrolyzing last-resort carbapenem antibiotics represent a major challenge for treatment of bacterial infections. Wide dissemination of these enzymes in Acinetobacter baumannii elevated this pathogen to the category of most deadly and difficult to treat. We present here the structure of the OXA-58 ß-lactamase, a major class D carbapenemase of A. baumannii, determined to 1.30-Å resolution. Unlike two other Acinetobacter carbapenemases, OXA23 and OXA-24, the OXA-58 enzyme lacks the characteristic hydrophobic bridge over the active site, despite conservation of the residues which participate in its formation. The active-site residues in OXA-58 are spatially conserved in comparison to those in other class D ß-lactamases. Lys86, which activates water molecules during the acylation and deacylation steps, is fully carboxylated in the OXA-58 structure. In the absence of a substrate, a water molecule is observed in the active site of the enzyme and is positioned in the pocket that is usually occupied by the 6α-hydroxyethyl moiety of carbapenems. A water molecule in this location would efficiently deacylate good substrates, such as the penicillins, but in the case of carbapenems, it would be expelled by the 6α-hydroxyethyl moiety of the antibiotics and a water from the surrounding medium would find its way to the vicinity of the carboxylated Lys86 to perform deacylation. Subtle differences in the position of this water in the acyl-enzyme complexes of class D ß-lactamases could ultimately be responsible for differences in the catalytic efficiencies of these enzymes against last-resort carbapenem antibiotics.

Class D ß-lactamases: are they all carbapenemases?

Carbapenem-hydrolyzing class D ß-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. The OXA-2 and OXA-10 enzymes predominantly occur in Pseudomonas aeruginosa and are currently classified as narrow-spectrum class D ß-lactamases. Here we demonstrate that when OXA-2 and OXA-10 are expressed in Escherichia coli strain JM83, they produce a narrow-spectrum antibiotic resistance pattern. When the enzymes are expressed in A. baumannii ATCC 17978, however, they behave as extended-spectrum ß-lactamases and confer resistance to carbapenem antibiotics. Kinetic studies of OXA-2 and OXA-10 with four carbapenems have demonstrated that their catalytic efficiencies with these antibiotics are in the same range as those of some recognized class D carbapenemases. These results are in disagreement with the classification of the OXA-2 and OXA-10 enzymes as narrow-spectrum ß-lactamases, and they suggest that other class D enzymes that are currently regarded as noncarbapenemases may in fact be CHDLs.

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. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4-16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M-1 s-1. Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k3 = (2.4 ± 0.3) × 10-7 s-1), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1-157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. These data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

Revisiting the nucleotide and aminoglycoside substrate specificity of the bifunctional aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia enzyme.

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Aminoglycoside 2''-phosphotransferase IIIa (APH(2'')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2'' kinases.

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2''-phosphotransferase IIIa (APH(2'')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2'')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.

Novel aminoglycoside 2''-phosphotransferase identified in a gram-negative pathogen.

Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6') acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6')-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (k(cat)/K(m) = 10(5) to 10(7) M(-1) s(-1)), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme.

A novel extended-spectrum ß-lactamase, SGM-1, from an environmental isolate of Sphingobium sp.

SGM-1 is a novel class A ß-lactamase from an environmental isolate of Sphingobium sp. containing all of the distinct amino acid motifs of class A ß-lactamases. It shares 77 to 80% amino acid sequence identity with putative ß-lactamases that are present on the chromosome of all Sphingobium species whose genomes were sequenced and annotated. Thus, SGM-type ß-lactamases are native to this genus. Antibiotic susceptibility testing classifies SGM-1 as an extended-spectrum ß-lactamase, conferring the highest level of resistance to penicillins. Although SGM-1 contains the conserved cysteine residues characteristic of class A carbapenemases, it does not confer resistance to the carbapenem antibiotics imipenem, meropenem, or doripenem but does increase the MIC of ertapenem 8-fold. SGM-1 hydrolyzes penicillins and the monobactam aztreonam with similar catalytic efficiencies, ranging from 10(5) to 10(6) M(-1) s(-1). The catalytic efficiencies of SGM-1 for cefoxitin and ceftazidime were the lowest (10(2) to 10(3) M(-1) s(-1)) among the cephalosporins tested, while the catalytic efficiencies against all other cephalosporins varied from about 10(5) to 10(6) M(-1) s(-1). SGM-1 exhibited measurable but not significant activity toward the carbapenems tested. SGM-1 also showed high affinity for clavulanic acid, tazobactam, and sulbactam (Ki < 1 µM); however, only clavulanic acid significantly reduced the MICs of ß-lactams.

Bulky "gatekeeper" residue changes the cosubstrate specificity of aminoglycoside 2''-phosphotransferase IIa.

The aminoglycoside 2"-phosphotransferases APH(2")-IIa and APH(2")-IVa can utilize ATP and GTP as cosubstrates, since both enzymes possess overlapping but discrete structural templates for ATP and GTP binding. APH(2″)-IIIa uses GTP exclusively, because its ATP-binding template is blocked by a bulky tyrosine "gatekeeper" residue. Replacement of the "gatekeeper" residues M85 and F95 in APH(2")-IIa and APH(2")-IVa, respectively, by tyrosine does not significantly change the antibiotic susceptibility profiles produced by the enzymes. In APH(2")-IIa, M85Y substitution results in an ~10-fold decrease in the K(m) value of GTP and an ~320-fold increase in the K(m) value of ATP. In APH(2")-IVa, F95Y substitution results in a modest decrease in the K(m) values of both GTP and ATP. Structural analysis indicates that in the APH(2")-IIa M85Y mutant, tyrosine blocks access of ATP to the correct position in the binding site, while the larger nucleoside triphosphate (NTP)-binding pocket of the APH(2")-IVa F95Y mutant allows the tyrosine to move away, thus giving access to the ATP-binding template.

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

The wide spread of carbapenem-hydrolyzing ß-lactamases in Gram-negative bacteria has diminished the utility of the last-resort carbapenem antibiotics, significantly narrowing the available therapeutic options. In the Enterobacteriaceae family, which includes many important clinical pathogens such as Klebsiella pneumoniae and Escherichia coli, production of class D ß-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. Here, we report evaluation of a novel, C5α-methyl-substituted carbapenem, NA-1-157, and show that its MICs against bacteria producing OXA-48-type enzymes were reduced by 4- to 32-fold when compared to meropenem. When combined with commercial carbapenems, the potency of NA-1-157 was further enhanced, resulting in target potentiation concentrations ranging from 0.125 to 2 µg/mL. Kinetic studies demonstrated that the compound is poorly hydrolyzed by OXA-48, with a catalytic efficiency 30- to 50-fold lower than those of imipenem and meropenem. Acylation of OXA-48 by NA-1-157 was severely impaired, with a rate 10,000- to 36,000-fold slower when compared to the commercial carbapenems. Docking, molecular dynamics, and structural studies demonstrated that the presence of the C5α-methyl group in NA-1-157 creates steric clashes within the active site, leading to differences in the position and the hydrogen-bonding pattern of the compound, which are incompatible with efficient acylation. This study demonstrates that NA-1-157 is a promising novel carbapenem for treatment of infections caused by OXA-48-producing bacterial pathogens.