The structure of the flavoenzyme glutathione reductase

Schulz, Georg E.; Schirmer, R. Heiner; Sachsenheimer, W.; Pai, Emil F.

doi:10.1038/273120a0

Cited by 279 publications

(173 citation statements)

References 22 publications

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“…As pointed out by Hofsteenge et al [5], the N-terminal region ofphydroxybenzoate hydroxylase has a characteristic AMPbinding pap-unit resembling that found in the mononucleotide-binding fold of the dehydrogenases; a PaB-unit was also proposed at the N terminus of D-amino acid oxidase (51. This structure is also present in glutathione reductase (both in the FAD-binding domain and the NADPH-binding domain) [30] ; more recently, similar sequences have been reported in corresponding areas of other flavoenzymes [32,37,38].…”

Section: Discussionmentioning

confidence: 94%

“…Moreover, the results add to the scarce structural information which existed thus far for the entire class of FAD-containing enzymes. Apart from p-hydroxybenzoate hydroxylase, other FAD-containing enzymes for which the primary and/or tertiary structure has been determined are human erythrocyte glutathione reductase (tertiary structure [30,31], primary structure [32,33]), pig kidney D-amino acid oxidase (primary structure [34]) and spinach ferredoxin -NADP+ oxidoreductase (tertiary structure [35,36]). The enzymes glutathione reductase, D-amino acid oxidase and p-hydroxybenzoate hydroxylase are representatives of three major classes of flavoproteins, viz.…”

Section: Discussionmentioning

confidence: 99%

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p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Weijer

Hofsteenge

Beintema

et al. 1983

European Journal of Biochemistry

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The complete primary and tertiary structure ofp-hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron-density map at 0.25-nm resolution. The parts of the polypeptide chain contributing the residues to the FAD-binding site and the residues of the substratebinding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p-hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD.Many side chains from the C-terminal part of the polypeptide chain are involved in subunit-subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.p-hydroxybenzoate hydroxylase from Pseudomonus ,fluerescens belongs to the class of flavin external monooxygenases. The enzyme catalyzes the incorporation of one atom from molecular oxygen in the substrate p-hydroxybenzoate with reduction by NADPH of the other oxygen atom. The enzyme forms dimers with two identical subunits. The monomer has a n~olecular weight of 43 x 1 O3 and contains no disulfide bridges [1,21.The three-dimensional structure of p-hydroxybenzoate hydroxylase with bound substrate has been determined at a resolution of 0.25 nm 131. In this complex the flavin is in the oxidized state and adopts a flat configuration. The entire FAD group is in an extended conformation and has numerous contacts with the protein. The polypeptide chain, which could be traced in the electron-density map, folds into three domains. Amino acids from all domains are involved in FAD binding. The substrate molecule is in close proximity to the isoalloxazine ring system. The enzyme contains much secondary structure, both a-helix (26%) and fi-sheet (16%). Moreover, the X-ray analysis confirms that the enzyme occurs as a dimer [3]. In the crystal the two subunits are related by a crystallographic twofold axis [3,4]. The contact area is formed by a domain predominantly built up from a-helices. The site of subunit interaction is well separated from the active site [3].In order to be able to interpret the electron-density map in terms of all the amino acid side chains, the primary structure of p-hydroxybenzoate hydroxylase was determined. The enzyme contains six methionines, one of which is N-terminal. After CNBr cleavage five peptides and free homoserine were isolated. The alignment of the CNBr fragments was deduced from the 0.25-nm electron-density map and sequence data. TheThe studies on peptide CB2 were part of the Ph. D. thesis of J. Hofsteenge presented in 1981 to the Rijksuniversiteit Groningen ; the studies on peptide CB1 will form part of the Ph. D. thesis of W. J. Weijer to be presented to the same university.Enzymes. p-Hydroxybenzoate hydroxylase (EC 1.14.13.2); glUtdthione reductdse (EC 1.6.4.2); D-amino-acid oxidase (EC 1.4.3.3); ferredoxin-NADP+ oxidoreductase (EC 1.1 8.1 2). __ -.-isolated f...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Weijer

Hofsteenge

Beintema

et al. 1983

European Journal of Biochemistry

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“…The observed dimer, burying a surface area of 3,260 Å 2 (38), involves the convex surface of each monomer. Dimers have also been reported for GR, PdxR, BphA4, and mAIF (19,20,23,33). In GR and similar S-S oxidoreductases, dimerization is essential as residues from both monomers create the active-site pocket (16,37).…”

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confidence: 99%

“…The fold of RdxR is related to those of GR (33), murine apoptosis inducing factor (mAIF) (20), the ferredoxin reductase component of biphenyl dioxygenase [bacterial ferredoxin reductase (BphA4)] (19,34), and putidaredoxin reductase (PdxR) (35) (see below). Although a sequence alignment of these proteins reveals several insertions and deletions in peripheral regions (SI Fig.…”

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confidence: 99%

Crystal structure of the electron transfer complex rubredoxin–rubredoxin reductase of Pseudomonas aeruginosa

Hagelueken

Wiehlmann

Adams

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gramnegative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for crosslinking, and both structures were refined at 2.40-and 2.45-Å resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 Å.

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“…In glutathione [24,25] the coenzyme appears to be in a similar conformation to that of NAD' in lactate dehydrogenase. The NADP+ in the dihydrofolate reductase ternary complex at 0.25-nm resolution is 0.3 nm more extended than NAD' when bound to the NAD+-dependent enzymes [26].…”

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confidence: 99%

Binding of Coenzyme and Substrate and Coenzyme Analogues to 6‐Phosphogluconate Dehydrogenase from Sheep Liver

Abdallah

Adams

Archibald

et al. 1979

European Journal of Biochemistry

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The analogues of the coenzyme NADP', nicotinamide-8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine -adenine dinucleotide phosphate (io3PdADP'), were prepared. Nbr'ADP' was found to be active in the hydrogen transfer and io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP', NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP' difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD' in the lactate dehydrogenase ternary complex. 6-Phosphogluconate dehydrogenase is an oxidative decarboxylase of the pentose phosphate pathway which catalyses the conversion of 6-phosphogluconate to ribulose 5-phosphate using the coenzyme NADP.The enzyme is dimeric with a monomer molecular weight of 50000. The two subunits act independently [l]. The mechanism of the oxidation has been studied under various conditions with different results. A random-order mechanism has been suggested in buffers such as Tris-acetate [2] while an ordered mechanism, binding of coenzyme followed by substrate, has been indicated in phosphate buffer at pH 7 [3]. Inhibition by various nucleotide phosphates, coenzyme fragments and other metabolic intermediates has been studied [4]. The coenzyme NAD' does not bind to the Abbreviations. ADP-Rib, adenosine diphosphoribose; io3PdAD(P)+, 3-iodopyridine -adenine dinucleotide (phosphate); n3PdAD(P)+, 3-aminopyridine ~ adenine dinucleotide (phosphate) ; Nbr*AD(P)+, nicotinamide -8-bromo-adenine dinucleotide (phosphate); NMR, nuclear magnetic resonance.Eniyme. 6-Phosphogluconate dehydrogenase (EC 1 .I .I .44).enzyme in solution [3]. A number of chemical modifications for this enzyme from several other sources have been described [5]. The sheep liver enzyme crystallizes in space group C 2221 (a = 7.27nm, b = 14.82nm, c = 10.29nm) with a single subunit in the asymmetric unit [6]. The 0.6-nm resolution structure of the enzyme has been determined using two heavy atom derivatives : potassium dicyanoaurate (I), KAU(CN)~, and potassium tetracyanoplatinate (11), KZPt(CN)4 [7]. The two subunits are related by a crystallographic twofold axis parallel to b. The subunit contains two a-helices more than 5....

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The structure of the flavoenzyme glutathione reductase

Cited by 279 publications

References 22 publications

p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Crystal structure of the electron transfer complex rubredoxin–rubredoxin reductase of Pseudomonas aeruginosa

Binding of Coenzyme and Substrate and Coenzyme Analogues to 6‐Phosphogluconate Dehydrogenase from Sheep Liver

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