Biochemical and structural characterization of Mycobacterium tuberculosis beta-lactamase with the carbapenems ertapenem and doripenem

Abstract

Despite the enormous success of beta-lactams as broad-spectrum antibacterials, they have never been widely used for the treatment of tuberculosis (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 beta-lactamase hydrolyzing all beta-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 conducted 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 provide apparent K(m) and k(cat) values of 0.18 microM and 0.016 min(-1) for doripenem and 0.18 microM and 0.017 min(-1) for ertapenem, respectively. FTICR mass spectrometry demonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The BlaC-doripenem covalent complex obtained after a 90 min soak was determined to 2.2 A, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 A. The 1.3 A diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original Delta(2)-pyrroline ring was tautomerized to generate the Delta(1)-pyrroline ring. The isomerization leads to the flipping of the carbapenem hydroxyethyl group to hydrogen bond to carboxyl O2 of Glu166. The hydroxyethyl flip results in both the decreased basicity of Glu166 and a significant increase in the distance between carboxyl O2 of Glu166 and the catalytic water molecule, slowing hydrolysis.

Figures

Figure 1

Time courses of doripenem (A) and ertapenem (B) hydrolysis with various concentrations of BlaC.

Figure 2

Time courses of nitrocefin hydrolysis by BlaC in the presence of doripenem (upper) and ertapenem (lower).

Figure 3

Mass spectra of enzyme-carbapenem species. The 25+ charge state ions are shown.

Figure 4

(A) Overall structure of BlaC displayed in rainbow from N term (blue) to the C term (red), with the doripenem adduct displayed in red surface mesh. (B) Fo-Fc omit density (green) contoured at 2.0 σ surrounds the covalent doripenem adduct formed at the Ambler active-site residue serine 70. All structure figures were produced using Pymol.

Figure 5

(A) Fo-Fc omit density (green) contoured at 2.0 σ surrounds the covalent ertapenem adduct formed at the Ambler active-site residue serine 70 in the pre-isomerization state. (B) Fo-Fc omit density (green) contoured at 2.0 σ surrounds the covalent ertapenem adduct formed at the Ambler active-site residue serine 70 in the post-isomerization state. The resolution of the densities unambiguously demonstrates the shift in stereochemistry with the change from sp2 to sp3 hybridization of the C3 carbapenem carbon atom with the change in the position of the density associated with the meta-amino-benzoate and the hydoxyethyl ertapenem moieties.

Scheme 1

(A) the structures of doripenem and ertapenem. (B) The chemical mechanism of hydrolysis of ertapenem by the Mycobacterium tuberculosis BlaC.