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

Abstract

BackgroundThe 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.FindingsWe 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 μM; wild-type = 29 μM) and NADPH (K69A = 30 μM; wild-type = 11 μM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4) μM and 134 (± 21), respectively.ConclusionOur 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.

Application of modified Michaelis – Menten equations for determination of enzyme inducing and inhibiting drugs

“…If the absorption is a limiting factor, terminal half-life is a reflection of rate and extent of absorption not elimination [ 60 ]. Also lysine 69 is catalytic via M. tuberculosis shikimate dehydrogenase and should be used in rational design of antitubercular drugs [ 97 ], perhaps with isoniazid. The Hill coefficient of 2.05 ± 0.1 could suggest multiple substrates binding site [ 98 ].…”

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

“…The mechanism of catalysis is not completely understood in detail. In agreement, two highly conserved active site residues, a lysine and an aspartate, were assigned an important role during catalysis (Lindner et al, 2005;Singh and Christendat, 2006;Bagautdinov and Kunishima, 2007;Gan et al, 2007;Rodrigues et al, 2009;Peek et al, 2011). Under debate is also a conserved tyrosine, which may stabilize the catalytic intermediate (Han et al, 2009), or a putative conserved water molecule in the active site, which might mediate the proton transfer to the bulk solvent (Bagautdinov and Kunishima, 2007;Gan et al, 2007).…”

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

“…The hydride transfer from NADPH C4 to the DHS hydroxyl group on C3 is mostly conducted by two well conserved Lys and Asp residues, while a conserved Tyr seems to be important to position the cofactor in a catalytic competent manner [90,95,96,98,104,105]. Site-directed mutagenesis data suggest an important role in catalysis for both residues, as an impact on k cat was observed in A. thaliana [98] and on M. tuberculosis [105]. Data from pH-rate profiles and isotope effects on MtSD also indicates that the deprotonation of a residue with pK a~8 .9 abolishes the activity of the enzyme [95].…”