Biochemical and structural characterization of mycobacterium tuberculosis β-lactamase with the carbapenems ertapenem and doripenem

Lee W. Tremblay, Fan Fan, John S. Blanchard

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Despite the enormous success of β-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 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 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 Km and kcat values of 0.18 μM and 0.016 min-1 for doripenem and 0.18 μM 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 Å, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 Å. The 1.3 Å diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original δ2-pyrroline ring was tautomerized to generate the δ1-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.

Original languageEnglish (US)
Pages (from-to)3766-3773
Number of pages8
JournalBiochemistry
Volume49
Issue number17
DOIs
StatePublished - May 4 2010

Fingerprint

doripenem
Carbapenems
Mycobacterium tuberculosis
Lactams
Isomerization
Tuberculosis
Enzymes
Alkalinity
Mass spectrometry
ertapenem
Hydrogen
Hydrolysis
Mass Spectrometry
Hydrogen bonds
Genes
Diffraction
Crystals
Molecules
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biochemical and structural characterization of mycobacterium tuberculosis β-lactamase with the carbapenems ertapenem and doripenem. / Tremblay, Lee W.; Fan, Fan; Blanchard, John S.

In: Biochemistry, Vol. 49, No. 17, 04.05.2010, p. 3766-3773.

Research output: Contribution to journalArticle

@article{7ad7c9cfc6914805af082d3309c2a085,
title = "Biochemical and structural characterization of mycobacterium tuberculosis β-lactamase with the carbapenems ertapenem and doripenem",
abstract = "Despite the enormous success of β-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 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 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 Km and kcat values of 0.18 μM and 0.016 min-1 for doripenem and 0.18 μM 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 {\AA}, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 {\AA}. The 1.3 {\AA} diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original δ2-pyrroline ring was tautomerized to generate the δ1-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.",
author = "Tremblay, {Lee W.} and Fan Fan and Blanchard, {John S.}",
year = "2010",
month = "5",
day = "4",
doi = "10.1021/bi100232q",
language = "English (US)",
volume = "49",
pages = "3766--3773",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - Biochemical and structural characterization of mycobacterium tuberculosis β-lactamase with the carbapenems ertapenem and doripenem

AU - Tremblay, Lee W.

AU - Fan, Fan

AU - Blanchard, John S.

PY - 2010/5/4

Y1 - 2010/5/4

N2 - Despite the enormous success of β-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 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 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 Km and kcat values of 0.18 μM and 0.016 min-1 for doripenem and 0.18 μM 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 Å, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 Å. The 1.3 Å diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original δ2-pyrroline ring was tautomerized to generate the δ1-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.

AB - Despite the enormous success of β-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 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 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 Km and kcat values of 0.18 μM and 0.016 min-1 for doripenem and 0.18 μM 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 Å, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 Å. The 1.3 Å diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original δ2-pyrroline ring was tautomerized to generate the δ1-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.

UR - http://www.scopus.com/inward/record.url?scp=77951676093&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77951676093&partnerID=8YFLogxK

U2 - 10.1021/bi100232q

DO - 10.1021/bi100232q

M3 - Article

VL - 49

SP - 3766

EP - 3773

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -