The structure of the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6min-1; R174A, 132.1min-1; R184A, 305.5min-1 versus wild type, 338.9min-1) and increased Km for NADPH (R144A, 5.3µM; R174A, 20.2µM; R184A, 54.4µM versus wild type, 3.9µM). The kcat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3min-1) and R184A (50.4min-1) compared with the wild type (33.0min-1), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2′-phosphate. Stopped-flow studies indicated that affinity (Kd) for NADPH was markedly reduced in mutants R144A (635µM) and R184A (2.3mM) compared with the wild type (< 5µM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH Kd ratio > 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (kred = 8.82s-1 versus 22.63s-1 for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2′-phosphate group, and R144 likely to interact with the nicotinamide ribose 5′-phosphate group.
Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase
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Claire LEADBEATER, Lisa MCIVER, Dominic J. CAMPOPIANO, Scott P. WEBSTER, Robert L. BAXTER, Sharon M. KELLY, Nicholas C. PRICE, Dominikus A. LYSEK, Michael A. NOBLE, Stephen K. CHAPMAN, Andrew W. MUNRO; Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase. Biochem J 1 December 2000; 352 (2): 257–266. doi: https://doi.org/10.1042/bj3520257
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