Bacterial Enzymes Interacting with β-Lactam Antibiotics

Georgopapadakou, Nafsika H.; Sykes, R. B.

doi:10.1007/978-3-642-68901-7_1

Cited by 19 publications

(14 citation statements)

References 253 publications

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“…Furthermore, the responses were noticeable at concentrations significantly below ( 1 / 16 th) the MIC and were dose-dependent, as can be seen in Figure a for E. coli exposed to amoxicillin. This observed differential response of E. coli cells to various β-lactam antibiotics is in agreement with the long-known differences in the target recognition profiles of this drug class . These results provided encouraging evidence that the use of machine learning could have a significant potential for the generation of classification models that allows the assessment of antibacterial activity and target identification.…”

Section: Resultssupporting

confidence: 85%

Machine Learning in Mass Spectrometry: A MALDI-TOF MS Approach to Phenotypic Antibacterial Screening

Oosten

Klein

2020

J. Med. Chem.

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Machine learning techniques can be applied to MALDI-TOF mass spectral data of drug-treated cells to obtain classification models which assign the mechanism of action of drugs. Here, we present an example application of this concept to the screening of antibacterial drugs that act at the major bacterial target sites such as the ribosome, penicillin-binding proteins, and topoisomerases in a pharmacologically relevant phenotypic setting. We show that antibacterial effects can be identified and classified in a label-free, high-throughput manner using wild-type Escherichia coli and Staphylococcus aureus cells at variable levels of target engagement. This phenotypic approach, which combines mass spectrometry and machine learning, therefore denoted as PhenoMS-ML, may prove useful for the identification and development of novel antibacterial compounds and other pharmacological agents.

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Section: Resultssupporting

confidence: 85%

Machine Learning in Mass Spectrometry: A MALDI-TOF MS Approach to Phenotypic Antibacterial Screening

Oosten

Klein

2020

J. Med. Chem.

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“…If this arrangement was the same in PBP 5 when bound to -lactam antibiotics, then the hydrolytic water must occupy a different position than O3 of the boronic acid inhibitor. More likely, however, is that PBP 5 binds lactams differently to the arrangement observed in PBP 2×, and the variety in binding (38). In this stereoview, the active-site residues of PBP 5 are shown as yellow bonds and those for the DD-peptidase are purple bonds.…”

Section: Structure Of Pbp 5 Bound To Boc-γ-d-glu-l-lys(cbz)-dboroalamentioning

confidence: 91%

Crystal Structure of Escherichia coli Penicillin-Binding Protein 5 Bound to a Tripeptide Boronic Acid Inhibitor: A Role for Ser-110 in Deacylation

et al. 2005

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Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.

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“…Penicillin‐binding proteins (PBPs) are ubiquitous bacterial enzymes that catalyze the last steps in cell wall biosynthesis (reviewed in [1–5]), and are the targets of the β‐lactam antibiotics. In Gram‐negative bacteria these enzymes catalyze the reactions shown in Scheme 1.…”

mentioning

confidence: 99%

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

Stefanova

Tomberg

Davies

et al. 2003

European Journal of Biochemistry

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The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the b-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for b-lactam binding, DD-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 M )1 AEs )1 ). Against a set of five a-and e-substituted L-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N e -acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N e -Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k 2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active sitedirected agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac 2 -L-Lys-D-Ala-D-Ala (k cat /K m ) was bell-shaped, with pK a values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs.

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Bacterial Enzymes Interacting with β-Lactam Antibiotics

Cited by 19 publications

References 253 publications

Machine Learning in Mass Spectrometry: A MALDI-TOF MS Approach to Phenotypic Antibacterial Screening

Machine Learning in Mass Spectrometry: A MALDI-TOF MS Approach to Phenotypic Antibacterial Screening

Crystal Structure of Escherichia coli Penicillin-Binding Protein 5 Bound to a Tripeptide Boronic Acid Inhibitor: A Role for Ser-110 in Deacylation

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

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