Probing the Determinants of Coenzyme Specificity in Ferredoxin-NADP+ Reductase by Site-directed Mutagenesis

Medina, Milagros; Luquita, Alejandra; Tejero, Jesús; Hermoso, J.A.; Mayoral, T.; Sanz‐Aparicio, Julia; Grever, Koert; Gómez‐Moreno, Carlos

doi:10.1074/jbc.m009287200

Cited by 58 publications

(99 citation statements)

References 60 publications

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“…We conclude from these data that Leu-76, Leu-78, and Val-136 of Anabaena FNR are not critical residues for complex formation and ET between NADPH and FNR. This was expected since the region where NADP(H) binds to FNR is different from the area where the mutations were introduced (24,50,51).…”

Section: Discussionmentioning

confidence: 99%

Role of a Cluster of Hydrophobic Residues Near the FAD Cofactor in Anabaena PCC 7119 Ferredoxin-NADP+Reductase for Optimal Complex Formation and Electron Transfer to Ferredoxin

Martínez‐Júlvez

Nogués

Faro

et al. 2001

Journal of Biological Chemistry

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In the ferredoxin-NADP؉ reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR⅐Fd complex.There are many examples throughout biology in which physiological function involves protein-protein interaction. Important illustrations of this are the reactions that occur between proteins in the ET 1 chains participating in photosynthesis, respiration, nitrogen fixation, and cytochrome P450 hydroxylations. In all of these cases, specific recognition and binding occur between the donor and the acceptor proteins. Although the structural parameters that determine such recognition are not fully understood, it is widely accepted that a transient complex between the two proteins must be formed and that the mutual orientation of the two proteins within the complex determines the rate of the ET reaction. For the last 10 years a large effort has gone into the study of the interaction between two ET proteins participating in the metabolism of the cyanobacterium Anabaena PCC 7119, ferredoxin (Fd), and ferredoxin-NADP ϩ reductase (FNR) (1-18). During photosynthesis, FNR catalyzes the transfer of electrons from the one-electron carrier Fd to the two-electron acceptor NADP ϩ to produce NADPH (19). Both proteins from Anabaena have been cloned and expressed in Escherichia coli (20 -22), and their high resolution x-ray structures have been determined (23,24). All of the biochemical and structural information available on these proteins indicates that Fd binds in a concave cavity around the FAD group of the reductase (2, 25), and this hypothesis has been strongly supported by the recent x-ray structures reported for the complex formed by Fd and FNR, either with the Anabaena or the maiz...

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Section: Discussionmentioning

confidence: 99%

Role of a Cluster of Hydrophobic Residues Near the FAD Cofactor in Anabaena PCC 7119 Ferredoxin-NADP+Reductase for Optimal Complex Formation and Electron Transfer to Ferredoxin

Martínez‐Júlvez

Nogués

Faro

et al. 2001

Journal of Biological Chemistry

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“…This structural and sequence similarity study (8) assigned PDR to a distinct family of flavoprotein reductases, all related to ferredoxin-NADP ϩ reductase (FNR). Medina et al (21) studied coenzyme specificity in FNR and found the amino acid residues involved. None of those residues are found in the reductase components shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Carbazole 1,9a-Dioxygenase, a Three-Component Dioxygenase System of Pseudomonas resinovorans Strain CA10

Nam

Nojiri

Noguchi

et al. 2002

Appl Environ Microbiol

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The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the k cat /K m for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.Pseudomonas resinovorans strain CA10 is a bacterium that has the ability to utilize carbazole (CAR) as its sole source of carbon, nitrogen, and energy. As the initial degradation reaction, CAR is dioxygenated at the angular (C-9a) and adjacent (C-1) positions to yield the unstable cis-hydrodiol (25, 27). The resultant cis-hydrodiol is spontaneously converted to 2Ј-aminobiphenyl-2,3-diol, which is further converted to anthranilate via meta cleavage and hydrolysis. Such initial dioxygenation is called angular dioxygenation and contains the degradation pathways of CAR and its structural analogues (25).Genes encoding CAR 1,9a-dioxygenase (CARDO) were cloned from P. resinovorans strain CA10, and CARDO was found to be a multicomponent enzyme system which consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd) (32). Amino acid sequence homology and phylogenetic analysis revealed that CarAa is a rather unique type of oxygenase that shares low homology with other known dioxygenases (23, 32). Furthermore, from studies on substrate specificity, it was found that CARDO has a broad substrate range and can catalyze diverse oxygenations: angular dioxygenation, lateral dioxygenation, and monooxygenation (26). In addition, Habe et al. (11) have reported that CARDO can attack some chlorinated dioxin congeners in a manner similar to that of dibenzofuran 4,4a-dioxygenase from Terrabacter sp. strain DBF63 and dioxin dioxygenase from Sphingomonas wittichii strain RW1 (43), although some difference was observed in particular features. Compared to other degradative reactions for dio...

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“…-4.2 (Liang et al 2007) P. stipitis XR 1K8C (h) K270R, N272D 2.9 9.3 -1.2 (Maddock et al 2015) E. coli CaADH 1KEV (h) G198D, S199V, P201E, Y218A >1 ? -4.6 (Maurer et al 2005) B Log Relative Activity c (Medina et al 2001) A. PCC7119 FDNR 2BSA S223D 0.12 8.1x10 3 -5.4 (Nakanishi et al 1997) M. musculus CR 1CYD T38D 31 1.3x10 3 -0.51 (Paladini et al 2009 (Schepens et al 2000) S. bicolor MDH 7MDH (1CIV) G84D, S85I, R87Q, S88A 12 2.1x10 4 -0.96 (Scrutton et al 1990) E. coli GTR 1GET A179G, A183G, V197E, R198M, K199F, H200D, R204P 8.1 1.8x10 4 -1.5 (Shiraishi et al 1998) N. crassa CbR S920D, R932S 65 7.2x10 4 -2.6 (Takase et al 2014) S. sp. A1-R 3AFN H37N, G38S, R39H, K40V, A41D >1 ?…”

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confidence: 99%

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

et al. 2016

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Probing the Determinants of Coenzyme Specificity in Ferredoxin-NADP+ Reductase by Site-directed Mutagenesis

Cited by 58 publications

References 60 publications

Role of a Cluster of Hydrophobic Residues Near the FAD Cofactor in Anabaena PCC 7119 Ferredoxin-NADP+Reductase for Optimal Complex Formation and Electron Transfer to Ferredoxin

Role of a Cluster of Hydrophobic Residues Near the FAD Cofactor in Anabaena PCC 7119 Ferredoxin-NADP+Reductase for Optimal Complex Formation and Electron Transfer to Ferredoxin

Purification and Characterization of Carbazole 1,9a-Dioxygenase, a Three-Component Dioxygenase System of Pseudomonas resinovorans Strain CA10

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

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