Alain Dubus

The roles of residues Tyr150, Glu272, and His314 in class C β‐lactamases

Serine β‐lactamases contribute widely to the β‐lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many “natural” and “artificial” mutants of these different enzymes have been isolated.

When Drug Inactivation Renders the Target Irrelevant to Antibiotic Resistance: A Case Story with Beta-Lactams

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a β‐lactamase‐overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor β‐lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents.

pKa calculations for class C β‐lactamases: The role of tyr‐150

The Poisson‐Boltzmann method was used to compute the pKa values of titratable residues in a set of class C β‐lactamases. In these calculations, the pKa of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pKa unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen‐bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pKa values were also computed in catalytically impaired β‐lactamase mutants. Comparisons between the relative kcat values and the Tyr150 pKa value in these mutants revealed a striking correlation. In active enzymes, this pKa value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed. Proteins 2000;40:23–28.

IND-6, a Highly Divergent IND-Type Metallo-β-Lactamase from Chryseobacterium indologenes Strain 597 Isolated in Burkina Faso

ABSTRACT The genus Chryseobacterium and other genera belonging to the family Flavobacteriaceae include organisms that can behave as human pathogens and are known to cause different kinds of infections. Several species of Flavobacteriaceae, including Chryseobacterium indologenes, are naturally resistant to β-lactam antibiotics (including carbapenems), due to the production of a resident metallo-β-lactamase. Although C. indologenes presently constitutes a limited clinical threat, the incidence of infections caused by this organism is increasing in some settings, where isolates that exhibit multidrug resistance phenotypes (including resistance to aminoglycosides and quinolones) have been detected. Here, we report the identification and characterization of a new IND-type variant from a C. indologenes isolate from Burkina Faso that is resistant to β-lactams and aminoglycosides. The levels of sequence identity of the new variant to other IND-type metallo-β-lactamases range between 72 and 90% (for IND-4 and IND-5, respectively). The purified enzyme exhibited N-terminal heterogeneity and a posttranslational modification consisting of the presence of a pyroglutamate residue at the N terminus. IND-6 shows a broad substrate profile, with overall higher turnover rates than IND-5 and higher activities than IND-2 and IND-5 against ceftazidime and cefepime.

Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay

Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no‐heme iron(II)‐dependent oxygenase which catalyses, in a single reaction, the bicyclisation of δ‐(L‐α‐aminoadipoyl)‐L‐cysteinyl‐D‐valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady‐state kinetic parameters for the IPNS reaction were determined.

Thiolester substrates of DD-peptidases and beta-lactamases

With peptide substrates, the penicillin-sensitive dd-peptidases exhibit a strict specificity for d-Ala-d-Xaa C-termini. Only glycine is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes also hydrolyse various ester and thiolester analogues of their natural substrates. Some of the thiolesters whose C-terminal leaving group exhibited an l stereochemistry were significantly hydrolysed by some of the studied enzymes, particularly by the Actinomadura R39 dd-peptidase. By contrast, the strict specificity for a d residue in the penultimate position was fully retained. The same esters and thiolesters also behaved as substrates for β-lactamases. In this case, thiolesters exhibiting l stereochemistry in the C-terminal position could also be hydrolysed, mainly by the class C and class D enzymes. But, more surprisingly, the class C Enterobacter cloacae P99 β-lactamase also hydrolysed thiolesters containing an l residue in the penultimate position, sometimes more efficiently than the d isomer.

Mechanism of action of β-lactamases and DD-peptidases

The introduction of penicillins as antibacterial agents probably represents one of the major breakthroughs of chemotherapy during the present century. However, the bacterial world did not remain without reaction and resistance to strains started to appear almost as soon as penicillin utilisation became popular. This resulted in an endless race between the chemists and the microbiologists on one side and the bacteria on the other, the formers discovering and synthesizing new compounds while the latters found new tricks to escape their lethal action so that the s-lactam family now comprises molecules of widely different structures of which the s-lactam ring remains the only common feature (Figure 1A).