Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Abstract

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Characterization of a class II ketol-acid reductoisomerase from Mycobacterium tuberculosis

“…The activity of MtKARI-II was estimated from NADPH oxidation by 2 by measuring the absorption at 340 nm using an extinction coefficient of 6220 M −1 cm −1 . By this assay, MtKARI-II was found to have catalytic parameters that are in good agreement with those reported for EcoKARI with k cat = 2.23 ± 0.1 s −1 , K M = 250 ± 30 μM, 18 MtKARI with k cat = 1.4 ± 0.02 s −1 , K M = 110 ± 4 μM (ref. 1 ) (Table S1 † ) and MtH37Rv (Mtb-Rv) and Mt H37Ra (Mtb-Ra) strains with a K M = 110 ± 4 μM.…”

“…By this assay, MtKARI-II was found to have catalytic parameters that are in good agreement with those reported for EcoKARI with S1 †) and MtH37Rv (Mtb-Rv) and Mt H37Ra (Mtb-Ra) strains with a K M ¼ 110 AE 4 mM. 18 MtKARI-II also displayed similar catalytic parameters to that found for KARIs from other microorganisms, suggesting a conserved reaction mechanism (Table S1 †). Interestingly, MtKARI-II could use NADH as the reducing agent, albeit less efficiently.…”

Phosphoglucomutase A mediated regulation of carbon flux is essential for antibiotic and disease persistence inMycobacterium tuberculosis

“…This together with our findings suggests that, despite showing a decrease in the transcript levels, pgmA is essential for the growth of bacteria under hypoxic conditions. Furthermore, our findings indicate that Phosphoglucomutase-β ( Rv3400 ), the protein of which has recently been studied 46 , did not show redundancy upon removal of pgmA .…”

Antibacterial Ingredients and Modes of the Methanol-Phase Extract from the Fruit of Amomum villosum Lour.

“…( p < 0.05). Of these, the ketol-acid reductoisomerase ( B4602_RS10775 ) was significantly repressed (0.447-fold) ( p < 0.05), which plays a crucial role in the biosynthesis pathway of branched-chain amino acids [ 22 ]. The 2-isopropylmalate synthase ( B4602_RS10780 ) was also significantly down-regulated (0.39-fold) ( p < 0.05), which catalyzes the first step of leucine biosynthesis [ 23 ].…”