Investigation of F420-dependent glucose-6-phosphate dehydrogenase variants using pH profiles and stopped-flow spectrometric ligand-binding methods

Abstract

**Please note that the full text is embargoed until 5/17/2024** ABSTRACT: F420-dependent glucose-6-phosphate dehydrogenase (FGD) from Mycobacteria tuberculosis, catalyzes a reaction the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone using the oxidized F420 cofactor, which is reduced to F420H2. The reaction is important within M. tuberculosis, the cause of tuberculosis disease (TB) due to its health relevance for treatment of multiple drug resistant and extreme drug resistant strains of M. tuberculosis. TB affects millions of people worldwide, and although treatable, multiple drug resistant and extreme drug resistant forms of TB adds strain on the current generation of drugs. Since this enzyme is not found in humans, is an ideal drug target. A past crystal structure of wild-type FGD from M. tuberculosis proposed that among the conserved residues, H40 and E109 function as the active site base and active site acid, respectively. In contrast, later pH profile studies on Glu109 and His 40 revealed that the while Glu109 acts as the acid, His 40 does not act as an active site base. Hence, our current focus is to determine which active site residue may act as active site base using the FGD variants of E13, H260 and H40. pH dependence studies were conducted to elucidate their roles in catalysis. The time-dependent binding experiments for wtFGD, E13, H40 were performed to get more mechanistic information such as koff and kon to incorporate into our global analyses. This work was conducted in collaboration with other group members and will be discussed here.