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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 69 and Cys 238 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 β-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 β-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

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.

The GES-2 β-lactamase is a class A carbapenemase, the emergence of which in clinically important bacterial pathogens is a disconcerting development as the enzyme confers resistance to carbapenem antibiotics. Tazobactam is a clinically used inhibitor of class A β-lactamases, which inhibits the GES-2 enzyme effectively, restoring susceptibility to β-lactam antibiotics. We have investigated the details of the mechanism of inhibition of the GES-2 enzyme by tazobactam. By the use of UV spectrometry, mass spectroscopy, and x-ray crystallography, we have documented and identified the involvement of a total of seven distinct GES-2·tazobactam complexes and one product of the hydrolysis of tazobactam that contribute to the inhibition profile. The x-ray structures for the GES-2 enzyme are for both the native (1.45 Å) and the inhibited complex with tazobactam (1.65 Å). This is the first such structure of a carbapenemase in complex with a clinically important β-lactam inhibitor, shedding light on the structural implications for the inhibition process.

A major mechanism of bacterial resistance to beta-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is the production of beta-lactamases. A handful of class A beta-lactamases have been discovered that have acquired the ability to turn over carbapenem antibiotics. This is a disconcerting development, as carbapenems are often considered last resort antibiotics in the treatment of difficult infections. The GES family of beta-lactamases constitutes a group of extended spectrum resistance enzymes that hydrolyze penicillins and cephalosporins avidly. A single amino acid substitution at position 170 has expanded the breadth of activity to include carbapenems. The basis for this expansion of activity is investigated in this first report of detailed steady-state and pre-steady-state kinetics of carbapenem hydrolysis, performed with a class A carbapenemase. Monitoring the turnover of imipenem (a carbapenem) by GES-1 (Gly-170) revealed the acylation step as rate-limiting. GES-2 (Asn-170) has an enhanced rate of acylation, compared with GES-1, and no longer has a single rate-determining step. Both the acylation and deacylation steps are of equal magnitude. GES-5 (Ser-170) exhibits an enhancement of the rate constant for acylation by a remarkable 5000-fold, whereby the enzyme acylation event is no longer rate-limiting. This carbapenemase exhibits k(cat)/K(m) of 3 x 10(5) m(-1)s(-1), which is sufficient for manifestation of resistance against imipenem.

Aminoglycoside 2''-phosphotransferases are clinically important enzymes that cause high levels of resistance to aminoglycoside antibiotics by the organisms that harbor them. These enzymes phosphorylate aminoglycosides, and the modified antibiotics show significant reduction in the binding ability to target the bacterial ribosome. This report presents a detailed characterization of the antibiotic resistance profile and the aminoglycoside and nucleotide triphosphate substrate profiles of four common aminoglycoside 2''-phosphotransferases widely distributed in clinically important Gram-positive microorganisms. Although the antibiotic resistance phenotypes exhibited by these enzymes are similar, their aminoglycoside and nucleotide triphosphate substrate profiles are distinctive. Contrary to the dogma that these enzymes use ATP as the source of phosphate in their reactions, two of the four aminoglycoside 2'-phosphotransferases utilize GTP as the phosphate donor. Of the other two enzymes, one exhibits preference for ATP, and the other can utilize either ATP or GTP as nucleotide triphosphate substrate. A new nomenclature for these enzymes is put forth that takes into account the differences among these enzymes based on their respective substrate preferences. These nucleotide triphosphate preferences should have ramifications for understanding of the evolution, selection, and dissemination of the genes for these important resistance enzymes.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created challenges in treatment of nosocomial infections. The recent clinical emergence of vancomycin-resistant MRSA is a new disconcerting chapter in the evolution of these strains. S. aureus normally produces four PBPs, which are susceptible to modification by beta-lactam antibiotics, an event that leads to bacterial death. The gene product of mecA from MRSA is a penicillin-binding protein (PBP) designated PBP 2a. PBP 2a is refractory to the action of all commercially available beta-lactam antibiotics. Furthermore, PBP 2a is capable of taking over the functions of the other PBPs of S. aureus in the face of the challenge by beta-lactam antibiotics. Three cephalosporins (compounds 1-3) have been studied herein, which show antibacterial activities against MRSA, including the clinically important vancomycin-resistant strains. These cephalosporins exhibit substantially smaller dissociation constants for the preacylation complex compared with the case of typical cephalosporins, but their pseudo-second-order rate constants for encounter with PBP 2a (k(2)/K(s)) are not very large (< or =200 m(-1) s(-1)). It is documented herein that these cephalosporins facilitate a conformational change in PBP 2a, a process that is enhanced in the presence of a synthetic surrogate for cell wall, resulting in increases in the k(2)/K(s) parameter and in more facile enzyme inhibition. These findings argue that the novel cephalosporins are able to co-opt interactions between PBP 2a and the cell wall in gaining access to the active site in the inhibition process, a set of events that leads to effective inhibition of PBP 2a and the attendant killing of the MRSA strains.

Aminoglycoside 3'-phosphotransferases (APH(3')s) are common bacterial resistance enzymes to aminoglycoside antibiotics. These enzymes transfer the gamma-phosphoryl group of ATP to the 3'-hydroxyl of the antibiotics, whereby the biological activity of the drugs is lost. Pre-steady-state and steady-state kinetics with two of these enzymes from Gram-negative bacteria, APH(3')-Ia and APH(3')-IIa, were performed. It is demonstrated that these enzymes in both ternary and binary complexes facilitate an ATP hydrolase activity (ATPase), which is competitive with the transfer of phosphate to the antibiotics. Because these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. Concentrations of the enzyme in vivo were determined, and it was estimated that in a single generation of bacterial growth there exists the potential that this activity would consume as much as severalfold of the total existing ATP. Studies with bacteria harboring the aph(3')-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3')-Ia gene, such that approximately 50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.

Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.

We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase. Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.