Summary
With the exception of HIV, tuberculosis (TB) is the leading cause of mortality among infectious diseases. The urgent need to develop new anti-tubercular drugs is apparent due to the increasing number of drug resistant Mycobacterium tuberculosis (Mtb) strains. Proteins involved in cholesterol import and metabolism have recently been discovered as potent targets against TB. FadA5, a thiolase from Mtb, is catalyzing the last step of the β-oxidation reaction of the cholesterol side-chain degradation under release of critical metabolites and was shown to be of importance during the chronic stage of TB infections. To gain structural and mechanistic insight on FadA5 we characterized the enzyme in different stages of the cleavage reaction and with a steroid bound to the binding pocket. Structural comparisons to human thiolases revealed that it should be possible to target FadA5 specifically and the steroid-bound structure provides a solid basis for the development of inhibitors against FadA5.
Mycobacterium
tuberculosis (Mtb), the causative agent
of tuberculosis (TB), is a highly successful human pathogen and has
infected approximately one-third of the world’s population.
Multiple drug resistant (MDR) and extensively drug resistant (XDR)
TB strains and coinfection with HIV have increased the challenges
of successfully treating this disease pandemic. The metabolism of
host cholesterol by Mtb is an important factor for
both its virulence and pathogenesis. In Mtb, the
cholesterol side chain is degraded through multiple cycles of β-oxidation
and FadA5 (Rv3546) catalyzes side chain thiolysis in the first two
cycles. Moreover, FadA5 is important during the chronic stage of infection
in a mouse model of Mtb infection. Here, we report
the redox control of FadA5 catalytic activity that results from reversible
disulfide bond formation between Cys59-Cys91 and Cys93-Cys377. Cys93
is the thiolytic nucleophile, and Cys377 is the general acid catalyst
for cleavage of the β-keto-acyl-CoA substrate. The disulfide
bond formed between the two catalytic residues Cys93 and Cys377 blocks
catalysis. The formation of the disulfide bonds is accompanied by
a large domain swap at the FadA5 dimer interface that serves to bring
Cys93 and Cys377 in close proximity for disulfide bond formation.
The catalytic activity of FadA5 has a midpoint potential of −220
mV, which is close to the Mtb mycothiol potential
in the activated macrophage. The redox profile of FadA5 suggests that
FadA5 is fully active when Mtb resides in the unactivated
macrophage to maximize flux into cholesterol catabolism. Upon activation
of the macrophage, the oxidative shift in the mycothiol potential
will decrease the thiolytic activity by 50%. Thus, the FadA5 midpoint
potential is poised to rapidly restrict cholesterol side chain degradation
in response to oxidative stress from the host macrophage environment.
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