The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

Abstract

Background: The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase.

Findings: We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 microM; wild-type = 29 microM) and NADPH (K69A = 30 microM; wild-type = 11 microM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (+/- 4) microM and 134 (+/- 21), respectively.

Conclusion: Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

Figure 1

The shikimate dehydrogenase-catalyzed reaction.

Figure 2

Steady-state kinetic measurements for wild-type (A and C) and K69A (B and D) MtbSD in the forward direction. A and B: DHS concentrations were varied while NADPH concentration was maintained at a fixed saturating level. C and D: NADPH concentrations were varied while DHS concentration was maintained at a fixed saturating level.

Figure 3

Spectroscopic measurements of intrinsic protein fluorescence for wild-type (black circles) and K69A (black squares) MtbSD upon DHS binding at varying concentrations.

Figure 4

MtbSD K69A model superimposed on experimentally solved T. thermophilus SD structure. Amino acid side chains involved in SHK binding and SHK molecule are shown as sticks. T. thermophilus and MtbSD K69A amino acids are colored, respectively, in light gray (residue number in bold) and dark gray (residue number in italics). H-bonds are shown as dotted lines; dashed lines represent H-bonds between Lys64/69, missing in MtbSD K69A model.