Role of Serine140 in the mode of action of Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA)

Abstract

Background: Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli.

Findings: Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data.

Conclusions: The results suggest that the main role of the S140 side chain of MabA is in catalysis. The S140 side chain appears to also play an indirect role in NADPH binding. Interestingly, NADPH titrations curves shifted from sigmoidal for WT to hyperbolic for S140A, suggesting that the S140 residue may play a role in displacing the pre-existing equilibrium between two forms of MabA in solution. The results here reported provide a better understanding of the mode of action of MabA that should be useful to guide the rational (function-based) design of inhibitors of MabA enzyme activity which, hopefully, could be used as lead compounds with anti-TB action.

Figure 1

Fluorescence spectroscopy of equilibrium binding of NADPH to MabA mutant proteins. Dependence of the enhancement in nucleotide fluorescence upon NADPH binding to S140A mutant. Inset: NADPH titration of S140T mutant protein.

Figure 2

LIGPLOT diagram for WT MabA and NADPH binary complex. Covalent bonds of NADPH are in purple, covalent bonds of amino acids in light brown, hydrogen bonds are in green dashed lines. Protein residues in hydrophobic contacts with NADPH are represented by red semi-circles with radiating spokes. There are sixteen hydrogen bonds between WT MabA and NADPH.

Figure 3

LIGPLOT for S140T MabA mutant and NADPH binary complex. Colour-coded bonds and intermolecular interactions are as for Figure 2. There are twelve hydrogen bonds between S140T MabA and NADPH.

Figure 4

LIGPLOT for S140A MabA mutant and NADPH binary complex. Colour-coded bonds and intermolecular interactions are as for Figure 2. There are ten hydrogen bonds between S140A MabA and NADPH.

Figure 5

The NADPH binding sites of WT MabA, S140A and S140T models are shown superimposed. The structures are represented in stick model. WT MabA (light gray), S140A (red), and S140T (blue).