The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

b-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to b-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 (Lys73) in the TEM-1 b-lactamase from Escherichia coli. These techniques included perturbation of the pKa of Lys73 by the study of the g-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 pKa of Lys73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu166 and Lys73 of class A b-lactamases has actually raised the pKa of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot definitively rule out that Glu166 activates the active site water, which in turn promotes Ser70 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 Glu166 and unprotonated Lys73. As such, unprotonated Lys73 could promote serine for acylation, a process that should be shared among all active-site-serine b-lactamases and penicillin-binding proteins

Cytoplasmic-Membrane Anchoring of a Class A β-Lactamase and Its Capacity in Manifesting Antibiotic Resistance▿

Bacterial β-lactamases are the major causes of resistance to β-lactam antibiotics. Three classes of these enzymes are believed to have evolved from ancestral penicillin-binding proteins (PBPs), enzymes responsible for bacterial cell wall biosynthesis. Both β-lactamases and PBPs are able to efficiently form acyl-enzyme species with β-lactam antibiotics. In contrast to β-lactamases, PBPs are unable to efficiently turn over antibiotics and therefore are susceptible to inhibition by β-lactam compounds. Although both PBPs and gram-negative β-lactamases operate in the periplasm, PBPs are anchored to the cytoplasmic membrane, but β-lactamases are not. It is believed that β-lactamases shed the membrane anchor in the course of evolution. The significance of this event remains unclear. In an attempt to demonstrate any potential influence of the membrane anchor on the overall biological consequences of β-lactamases, we fused the TEM-1 β-lactamase to the C-terminal membrane-anchor of penicillin-binding protein 5 (PBP5) of Escherichia coli. The enzyme was shown to express well in E. coli and was anchored to the cytoplasmic membrane. Expression of the anchored enzyme did not result in any changes in antibiotic resistance pattern of bacteria or growth rates. However, in the process of longer coincubation, the organism that harbored the plasmid for the anchored TEM-1 β-lactamase lost out to the organism transformed by the plasmid for the nonanchored enzyme over a period of 8 days of continuous growth. The effect would appear to be selection of a variant that eliminates the problematic protein through elimination of the plasmid that encodes it and not structural or catalytic effects at the protein level. It is conceivable that an evolutionary outcome could be the shedding of the sequence for the membrane anchor or alternatively evolution of these enzymes from nonanchored progenitors

Purification, crystallization and preliminary X-ray analysis of the β-lactamase Oih-1 from Oceanobacillus iheyensis

Oih-1, a β-lactamase enzyme isolated from the deep-sea bacterium O. iheyensis, has been crystallized and a complete X-ray diffraction data set has been collected to 1.65 Å resolution

Intrinsic Class D β-Lactamases of Clostridium difficile

C. difficile is a spore-forming anaerobic bacterium which causes infection of the large intestine with high mortality rates. The C. difficile infection is difficult to prevent and treat, as the pathogen is resistant to many antimicrobial agents. Prolonged use of β-lactam antibiotics for treatment of various infectious diseases triggers the infection, as these drugs suppress the abundance of protective gut bacteria, allowing the resistant C. difficile bacteria to multiply. While resistance of C. difficile to β-lactam antibiotics plays the major role in the development of the disease, the mechanism of resistance is unknown. The significance of our research is in the discovery in C. difficile of β-lactamases, enzymes that destroy β-lactam antibiotics. These findings ultimately can help to combat deadly C. difficile infections.Clostridium difficile is the causative agent of the deadly C. difficile 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 C. difficile encodes class D β-lactamases, i.e., CDDs, which are intrinsic to this species. We studied two CDD enzymes, CDD-1 and CDD-2, and showed that they display broad-spectrum, high catalytic efficiency against various β-lactam antibiotics, including penicillins and expanded-spectrum cephalosporins. We demonstrated that the cdd genes are poorly expressed under the control of their own promoters and contribute only partially to the observed resistance to β-lactams. However, when the cdd1 gene was expressed under the control of efficient promoters in the antibiotic-sensitive Clostridium cochlearium strain, it produced high-level resistance to β-lactams. Taken together, the results determined in this work demonstrate the existence in C. difficile of intrinsic class D β-lactamases which constitute a reservoir of highly potent enzymes capable of conferring broad-spectrum, clinically relevant levels of resistance to β-lactam antibiotics. This discovery is a significant contribution to elucidation of the mechanism(s) of resistance of the clinically important pathogen C. difficile to β-lactam antibiotics

Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal

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 AAC(6’)-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6’ group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6’)-Im, thus explaining the lack of resistance to this molecule

Importance of Position 170 in the Inhibition of GES-Type β-Lactamases by Clavulanic Acid▿

Bacterial resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is commonly the result of the production of β-lactamases. The emergence of β-lactamases capable of turning over carbapenem antibiotics is of great concern, since these are often considered the last resort antibiotics in the treatment of life-threatening infections. β-Lactamases of the GES family are extended-spectrum enzymes that include members that have acquired carbapenemase activity through a single amino acid substitution at position 170. We investigated inhibition of the GES-1, -2, and -5 β-lactamases by the clinically important β-lactamase inhibitor clavulanic acid. While GES-1 and -5 are susceptible to inhibition by clavulanic acid, GES-2 shows the greatest susceptibility. This is the only variant to possess the canonical asparagine at position 170. The enzyme with asparagine, as opposed to glycine (GES-1) or serine (GES-5), then leads to a higher affinity for clavulanic acid (Ki = 5 μM), a higher rate constant for inhibition, and a lower partition ratio (r ≈ 20). Asparagine at position 170 also results in the formation of stable complexes, such as a cross-linked species and a hydrated aldehyde. In contrast, serine at position 170 leads to formation of a long-lived trans-enamine species. These studies provide new insight into the importance of the residue at position 170 in determining the susceptibility of GES enzymes to clavulanic acid