Abstractβ-lactam antibiotics are ineffective against Mycobacterium tuberculosis, being rapidly hydrolyzed by the chromosomally encoded blaC gene product. The carbapenem class of β-lactams are very poor substrates for BlaC, allowing us to determine the three-dimensional structure of the covalent BlaCmeropenem covalent complex at 1.8 angstrom resolution. When meropenem was combined with the β-lactamase inhibitor clavulanate, potent activity against laboratory strains of M. tuberculosis was observed [minimum inhibitory concentration (MIC meropenem ) less than 1 microgram per milliliter], and sterilization of aerobically grown cultures was observed within 14 days. In addition, this combination exhibited inhibitory activity against anaerobically grown cultures that mimic the "persistent" state and inhibited the growth of 13 extensively drug-resistant strains of M. tuberculosis at the same levels seen for drug-susceptible strains. Meropenem and clavulanate are Food and Drug Administration-approved drugs and could potentially be used to treat patients with currently untreatable disease.
Despite the enormous success of β-lactams as broad-spectrum antibacterials, they have never been widely used for the treatment of TB due to intrinsic resistance that is caused by the presence of a chromosomally-encoded gene (blaC) in Mycobacterium tuberculosis. Our previous studies of TB BlaC revealed that this enzyme is an extremely broad-spectrum β-lactamase hydrolyzing all β-lactam classes. Carbapenems are slow substrates that acylate the enzyme but are only slowly deacylated and can therefore act also as potent inhibitors of BlaC. We carried out the in vitro characterization of doripenem and ertapenem with BlaC. A steady-state kinetic burst was observed with both compounds with magnitudes proportional to the concentration of BlaC used. The results show apparent K m and k cat values of 0.18 µM and 0.016 min −1 for doripenem and 0.18 µM and 0.017 min −1 for ertapenem. FTICR mass spectrometry demonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The BlaCdoripenem covalent complex obtained after 90 minutes of soaking was solved to 2.2 Å, while the BlaC-ertapenem complex obtained after a 90 minute soak was solved to 2.0 Å. The 1.3 Å diffraction data from a 10 minute ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original Δ 2 pyrroline ring was tautomerized to generate the Δ 1 pyrroline ring. The isomerization leads to the flipping of the carbapenem-hydroxyethyl group to hydrogen bond to the carboxyl O2 of Glu166. The hydroxyethyl flip results in both decreased basicity of Glu166 and in a significant increase in the distance between the carboxyl O2 of Glu166 and the catalytic water molecule, slowing hydrolysis. Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a worldwide health concern (1). There were an estimated 9.3 million new cases of TB in 2007 and approximately 1.3 million HIV-negative patient fatalities as well as nearly half a million deaths amongst HIV-positive populations (2). Even fifty years after the introduction of powerful antibiotics to treat TB, it has been estimated that one person is infected in the world every few seconds (3). The failure to control TB is due to the emergence of M. tuberculosis strains that are multiply drug resistant towards the front line antimycobacterial drugs such as isoniazid and rifampicin.Phone: (718) Fax: (718) As one of the most important antibiotic families, β-lactams include a broad range of molecules including penicillin derivatives, cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors. The carbapenems exhibit the broadest spectrum of activity among the β-lactam antimicrobials, providing safe and efficacious therapies in the treatment of serious infections caused by Gram-positive, Gram-negative, and anaerobic bacterial pathogens (4,5). Carbapenem antibiotics were originally developed from thienamycin, a natural product identified in culture filtrates of Streptomyces cattleya (6). There are four carbapen...
Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.
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