Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis

Abstract

F420H2:NADP+ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP+ to NADPH by transferring a hydride from the reduced F420 cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP+ and the F420 precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased kcat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs420 versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s−1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH2 to NADP+. Therefore, Fno is a dynamic enzyme that regulates NADPH production.

Fop

“…This positive cooperativity can be explained by the dimeric structure of the enzyme. Cooperativity was also observed for the FNO from Archaeoglobus fulgidus, using NADPH and FO, as reported by Le et al 274 Strangely, positive cooperativity was not seen with F 420 as a coenzyme, as discussed by Kumar and Nguyen et al 253 The kinetic parameters for FO and FOP, however, are similar to that of F 420 (Figure 3.1). Thus, FO, FOP and F 420 are all equally well used as coenzymes in NADPH oxidation by TfuFNO.…”

Chemoenzymatic Synthesis of an Unnatural Deazaflavin Cofactor That Can Fuel F420-Dependent Enzymes

“…This positive cooperativity can be explained by the dimeric structure of the enzyme. Cooperativity was also observed for the FNO from Archaeoglobus fulgidus , using NADPH and FO, as reported by Le et al Strangely, positive cooperativity was not seen with F 420 as a coenzyme, as discussed by Kumar and Nguyen et al The kinetic parameters for FO and FOP, however, are similar to that of F 420 (see Table ). Thus, FO, FOP and F 420 are all equally well used as coenzymes in NADPH oxidation by TfuFNO.…”

Structure/activity virtual screening and in vitro testing of small molecule inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from gut methanogenic bacteria

“…Here, the spectrofluorometric assay of FNO activity was based on monitoring the decrease in F420 emission at 470 nm upon excitation at 420 nm. Briefly, FNO (4 nM) was incubated with 0.5 mM F420 (purified from T. roseum as described in the Supplementary Information) and 0.5 mM NADPH (this concentration was selected on the basis of K m values in other methanogenic organisms 42 ) at 37 °C in the presence or in the absence of increasing concentrations of the compounds of interest in 50 mM sodium citrate, pH 6.0. The apparent dissociation constant ( K D,app ) of the preformed FNO- ligand complex on the NADPH binding site was determined by measuring the decrease in the catalytic activity upon addition of increasing levels of the ligands (baicalin 0–140 μM; mangiferin 0–3.1 mM; β-D-glucose pentaacetate 0–7.2 mM; frangulin A 0–820 μM; ononin 0–950 μM; 5z-caffeoylquinic acid 0–840 µM; ZINC35442308 0–800 µM; ZINC248099510 0–840 µM; ZINC2120951 0–830 µM; ZINC9271779 0–830 µM).…”