Rkr. Voladri et al., RECOMBINANT EXPRESSION AND CHARACTERIZATION OF THE MAJOR BETA-LACTAMASE OF MYCOBACTERIUM-TUBERCULOSIS, Antimicrobial agents and chemotherapy, 42(6), 1998, pp. 1375-1381
New antibiotic regimens are needed for the treatment of multidrug-resi
stant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglyc
an layer, and the penicillin-binding proteins involved in its biosynth
esis are inhibited by clinically relevant concentrations of beta-lacta
m antibiotics. beta-Lactamase production appears to be the major mecha
nism by which M. tuberculosis expresses beta-lactam resistance. beta-L
actamases from the broth supernatant of 3- to 4-week-old cultures of h
. tuberculosis H37Ra were partially purified by sequential gel filtrat
ion chromatography and chromatofocusing. Three peaks of beta-lactamase
activity with pi values of 5.1, 4.9, and 4.5, respectively, and which
accounted for 10, 78, and 12% of the total postchromatofocusing beta-
lactamase activity, respectively, were identified. The beta-lactamases
with pi values of 5.1 and 4.9 were kinetically indistinguishable and
exhibited predominant penicillinase activity. In contrast, the beta-la
ctamase with a pi value of 4.5 showed relatively greater cephalosporin
ase activity. An open reading frame in cosmid Y49 of the DNA library o
f M. tuberculosis H37Rv with homolog to known class A beta-lactamases
was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and
was overexpressed in Escherichia coli. The recombinant enzyme was kin
etically similar to the pi 5.1 and 4.9 enzymes purified directly from
M. tuberculosis, It exhibited predominant penicillinase activity and w
as especially active against azlocillin. It was inhibited by clavulani
c acid and m-aminophenylboronic acid but not by EDTA. We conclude that
the major beta-lactamase of M. tuberculosis is a class A beta-lactama
se with predominant penicillinase activity. A second, minor beta-lacta
mase with relatively greater cephalosporinase activity is also present
.