Biochemical and Structural Characterization of Mycobacterium tuberculosis β-Lactamase with the Carbapenems Ertapenem and Doripenem

摘要

Despite the enormous success of β-lactams as broad-spectrumantibacterials, they have never beennwidely used for the treatment of tuberculosis (TB) due to intrinsic resistance that is caused by the presence of anchromosomally encoded gene (blaC)inMycobacteriumtuberculosis.Our previous studies of TB BlaC revealednthat this enzyme is an extremely broad-spectrum β-lactamase hydrolyzing all β-lactam classes. Carbapenemsnare slow substrates that acylate the enzyme but are only slowly deacylated and can therefore act also as potentninhibitors of BlaC. We conducted the in vitro characterization of doripenem and ertapenem with BlaC. Ansteady-state kinetic burst was observed with both compounds with magnitudes proportional to thenconcentration of BlaC used. The results provide apparent Km and kcat values of 0.18 μM and 0.016 min-1nfor doripenem and 0.18 μM and 0.017 min-1nfor ertapenem, respectively. FTICR mass spectrometryndemonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time periodnof 90 min. The BlaC-doripenem covalent complex obtained after a 90 min soak was determined to 2.2 A,nwhile the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 A.The1.3Andiffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring innthe BlaC-ertapenem adduct in which the original Δ2n-pyrroline ring was tautomerized to generate thenΔ1n-pyrroline ring. The isomerization leads to the flipping of the carbapenem hydroxyethyl group tonhydrogen bond to carboxyl O2 of Glu166. The hydroxyethyl flip results in both the decreased basicity ofnGlu166 and a significant increase in the distance between carboxyl O2 of Glu166 and the catalytic waternmolecule, slowing hydrolysis.