Kinetics and Binding Studies Reveal that His‐40 is Essential for Catalysis in F420‐Dependent Glucose‐6‐Phosphate Dehydrogenase from Mycobacterium tuberculosis

Abstract

Tuberculosis disease (TB) is a deadly infectious disease that is caused by Mycobacterium tuberculosis (Mtb). Our study focuses on F420‐dependent glucose‐6‐phosphate dehydrogenase (FGD), the enzyme that catalyzes the first committed step of the pentose phosphate pathway within Mtb. FGD uses the oxidized F420 cofactor to convert glucose‐6‐phosphate (G6P) to 6‐phosphogluconolactone, yielding reduced cofactor F420H2. F420H2 has been linked to the activation of a promising TB pro‐drug (PA‐824) and it also protects Mtb from nitrosative damages caused by the host's macrophage system. The FGD crystal structure revealed an active site His‐40 residue that has been proposed to be the active site base for the FGD reaction. We aim to probe the functionality of this His‐40 residue using site‐directed mutagenesis, crystallography, binding assays, circular dichroism, and steady state kinetic methods. Here, we present the results of the characterization of wild‐type FGD (wtFGD) and one FGD His‐40 mutant (FGD H40A). The circular dichroism data showed similar spectra for the FGD H40A and wtFGD, suggesting that the H40A mutation did not cause any structural changes within the enzyme. The steady state kinetics yielded kcat values of 1.40 ± 0.03 s−1 for wtFGD and 0.0016 ± 0.00 s−1 for FGD H40A, thus resulting in over 1000‐fold decrease in catalytic efficiency for the FGD H40A mutant. From the binding assays, we were able to conclude that His‐40 is not involved in G6P binding, but it helps in anchoring the F420 cofactor into the active site. The drastic loss in catalytic efficiency for the FGD H40A mutant led to crystallography experiments aimed at solving an FGD crystal structure with both F420 and G6P in the active site. FGD H40A crystals grew in 0.1 M ammonium citrate dibasic (pH 5.0) with 19% PEG 3350 as the precipitant, and diffracted to 2.9 Å to reveal the basis of substrate binding. In conclusion, the results presented here support the previously proposed mechanism which suggested that His‐40 is the active site base for the FGD reaction.Support or Funding InformationThis research was supported by NSF Grants 1120837