Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Leadbeater, Claire; McIver, Lisa; Campopiano, Dominic J.; Webster, Scott P.; Baxter, Robert L.; Kelly, Sharon M.; Price, Nicholas C.; Lysek, D.A.; Noble, Michael A.; Chapman, Stephen K; Munro, Andrew W.

doi:10.1042/bj3520257

Cited by 17 publications

(14 citation statements)

References 31 publications

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“…In the near‐UV region, all FNRs exhibited very strong, sharp positive and negative signals at 271 and 286 nm, respectively. A similar strong signal at 272 nm, observed in the CD spectrum of E. coli Fld oxidoreductase, has been attributed to the stacked interactions between FAD and one or more aromatic residues [27]. The introduced mutations in FNR did not alter the position of this near‐UV band.…”

Section: Resultssupporting

confidence: 55%

Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

et al. 2008

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Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs, EC 1.18.1.2) are a widely distributed class of flavoenzymes that have non-covalently bound FAD cofactor as a redox center. FNRs participate in a wide variety of redox-based metabolic reactions, transferring electrons between obligatory one-and two-electron carriers and therefore functioning as a general electron splitter. In non-phototrophic bacteria and eukaryotes, the reaction is driven towards ferredoxin (Fd) reduction, providing reducing power for multiple metabolic pathways, including steroid hydroxylation in mammalian mitochondria, nitrite reduction and glutamate synthesis in heterotrophic tissues of vascular plants, radical propagation and scavenging in prokaryotes, and hydrogen and nitrogen fixation in anaerobes (for a review, see [1,2]). In plants, FNR participates in photosynthetic electron transport, reducing Fd at the level of photosystem I, and transferring electrons to NADP + . This process ends with the formation of the NADPH necessary for CO 2 fixation and other biosynthetic pathways [2].The three-dimensional structures of several FNRs have been determined. They display similar structural features, which have been defined as the prototype for a large family of flavoenzymes [3][4][5][6][7][8][9][10] Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases. In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20-to 100-fold lower than those of their plastidic and cyanobacterial counterparts. Several structural features of these enzymes have yet to be explained. The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the flavin and is highly conserved amongst members of the family. By computing the relative free energy for the lumiflavin-phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine. This effect is probably caused by amino acids C266 and L268, which face the other side of this tyrosine. Simple and double FNR mutants of these amino acids were obtained and characterized. It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure. Our results provide experimental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme.

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Section: Resultssupporting

confidence: 55%

Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

et al. 2008

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“…The rates of individual electron transfer steps are consistent with this slow pace of catalysis ([62,63], summarized in Table 2). Moreover, E. coli FNR mediates direct reduction of cytochrome c at ≈ 5 s −1 , with this rate being enhanced only about twofold by the addition of saturating flavodoxin [62]. Similar low activities have been obtained with the A. vinelandii [74] and Rhodobacter capsulatus (C. Bittel, N. Carrillo and N. Cortez, IBR, Rosario, Argentina, unpublished observations) reductases.…”

Section: The Backward Reaction Is a Mechanistic Puzzlesupporting

confidence: 55%

“…This reductase species is remarkably slow, turning over at 0.15 s −1 for Fd and about 0.004 s −1 for the flavodoxins [63]. The rates of individual electron transfer steps are consistent with this slow pace of catalysis ([62,63], summarized in Table 2). Moreover, E. coli FNR mediates direct reduction of cytochrome c at ≈ 5 s −1 , with this rate being enhanced only about twofold by the addition of saturating flavodoxin [62].…”

Section: The Backward Reaction Is a Mechanistic Puzzlementioning

confidence: 65%

“…This thermodynamically unfavoured process results in a decrease of the binding affinity for NADP(H) relative to those of the 2′‐P‐AMP and 2′‐P‐ADP‐ribose analogues [61]. A similar movement has been postulated for the penultimate tyrosine residue of E. coli FNR, although in this case the carboxyl terminal tryptophan interacting with the adenosine moiety of FAD might also undergo a conformational change upon substrate binding [32,62–64].…”

Section: Nadp(h) Binding (Fig 2 Steps 1 and 9)mentioning

confidence: 99%

“…7B). It has been argued that the low rate of hydride transfer displayed by E. coli FNR is caused by the stacking interactions between Tyr247 and Trp248 with the isoalloxazine and adenine ring systems, that are expected to shield the NADP(H) binding site and impede easy access of the cofactor [62–64]. However, the affinity for the pyridine nucleotide is actually higher in the bacterial FNR (Table 2).…”

Section: Open Question: the Best Possible Ferredoxin‐nadp(h) Reductasementioning

confidence: 99%

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Open questions in ferredoxin‐NADP⁺ reductase catalytic mechanism

Carrillo

Ceccarelli

2003

European Journal of Biochemistry

244

290

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Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP + or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.

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Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners

Ugalde

Koning

Wallraven

et al. 2018

Appl Microbiol Biotechnol

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Mycobacterium tuberculosis (Mtb) codes for 20 cytochrome P450 enzymes (CYPs), considered potential drug-targets due to their essential roles in bacterial viability and host infection. Catalytic activity of mycobacterial CYPs is dependent on electron transfer from a NAD (P)H-ferredoxin-reductase (FNR) and a ferredoxin (Fd). Two FNRs (FdrA and FprA) and five ferredoxins (Fdx, FdxA, FdxC, FdxD, and Rv1786) have been found in the Mtb genome. However, as of yet, the cognate redox partnerships have not been fully established. This is confounded by the fact that heterologous redox partners are routinely used to reconstitute Mtb CYP metabolism. To this end, this study aimed to biochemically characterize and identify cognate redox partnerships for Mtb CYPs. Interestingly, all combinations of FNRs and ferredoxins were active in the reduction of oxidized cytochrome c, but steady-state kinetic assays revealed FdxD as the most efficient redox partner for FdrA, whereas Fdx coupled preferably with FprA. CYP121A1, CYP124A1, CYP125A1, and CYP142A1 metabolism with the cognate redox partners was reconstituted in vitro showing an unanticipated selectivity in the requirement for electron transfer partnership, which did not necessarily correlate with proximity in the genome. This is the first description of microbial P450 metabolism in which multiple ferredoxins are functionally linked to multiple CYPs.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9299-4) contains supplementary material, which is available to authorized users.

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Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Cited by 17 publications

References 31 publications

Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

Open questions in ferredoxin‐NADP⁺ reductase catalytic mechanism

Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners

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Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Cited by 17 publications

References 31 publications

Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

Open questions in ferredoxin‐NADP+ reductase catalytic mechanism

Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners

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Open questions in ferredoxin‐NADP⁺ reductase catalytic mechanism