A new catalytic mechanism of bacterial ferredoxin‐NADP⁺ reductases due to a particular NADP⁺ binding mode

Abstract

Ferredoxin-NADP + reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant-and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes. The plastidic FNRs show turnover numbers between 20 and 100 times higher than bacterial enzymes and these differences have been related to their physiological functions. We demonstrated that purified Escherichia coli FPR (EcFPR) contains tightly bound NADP + , which does not occur in plastidic type FNRs. The three-dimensional structure of EcFPR evidenced that NADP + interacts with three arginines (R144, R174, and R184) which could generate a very high affinity and structured site. These arginines are conserved in other bacterial FNRs but not in the plastidic enzymes. We have cross-substituted EcFPR arginines with residues present in analogous positions in the Pisum sativum FNR (PsFNR) and replaced these amino acids by arginines in PsFNR. We analyzed all proteins by structural, kinetic, and stability studies. We found that EcFPR mutants do not contain bound NADP + and showed increased K m for this nucleotide. The EcFPR activity was inhibited by NADP + but this behavior disappeared as arginines were removed. A NADP + analog of the nicotinamide portion produced an activating effect on EcFPR and promoted the NADP + release. Our results give evidence for a new model of NADP + binding and catalysis in bacterial FNRs.We propose that this tight NADP + binding constitutes an essential catalytic and regulatory mechanism of bacterial FNRs involved in redox homeostasis. K E Y W O R D S catalytic mechanism, catalytic site, Escherichia coli, ferredoxin-NADP + reductase, NADP + binding 1 | INTRODUCTION Ferredoxin-NADP + reductases (FNRs) constitute a family of monomeric hydrophilic proteins that contain noncovalently bound FAD as a prosthetic group. These enzymes are widely distributed among organisms and are

Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP+

“…Beyond the specific information regarding the dissociation of AnFNR ox :NADP + complexes and the role of Y303 in blocking nicotinamide access to the access site, this study paves the way to use DFS by AFM to characterize, at the single-molecule level, the occurrence and relevance of bipartite binding sites in pyridine nucleotide-dependent enzymes. In line with this, by considering plastidic and bacterial FNRs, we can envision different profiles among family members with different adaptation of their active sites to divergent dynamics and organizations during catalysis, with recognition mechanisms that either favor the initial binding of either of the two nucleotide moieties of the coenzyme [55,56]. To complete the study of the interactions in this electron transfer chain, it would be desirable to operate DFS also by preserving the FAD cofactor and the NADP + substrate, in the oxidized and in the reduced state, respectively.…”

NADP(H)-dependent biocatalysis without adding NADP(H)

“…S1 A and B ) ( 14 , 19 – 22 ). Other proteins that copurify with bound NAD(H) ( 23 – 32 ) and NADP(H) ( 33 – 39 ) include the nicotinoproteins ( 28 – 32 , 37 , 38 , 40 ) that apparently use NAD(P)(H) as a permanently bound prosthetic group (instead of an exchangeable cofactor) to perform a variety of reactions. The limited examples of NAD(P)(H)-dependent dehydrogenases copurifying with nicotinamide cofactors ( 23 , 24 , 32 – 35 ) suggest that extremely tight cofactor binding may be unusual.…”

Motivation toward Physical Activity and Healthy Habits of Adolescents: A Systematic Review

“…Similarly, students with a higher level of regular PA practice registered more intrinsic motivation and identified regulation towards PE and lower motivation in those who do not practice DFA. Other personal factors, such as sport self-concept, physical self-concept or perceived competence, are also key predictors of future PA [ 51 , 52 , 53 ]. It is also observed how factors related to self-perception and those that were most frequently associated with enjoyment and potential health benefits become motivational factors that influence self-determination to perform PA among adolescents.…”