Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site

Fischer, Katrin; Barbier, Guillaume G.; Hecht, H.J.; Mendel, Ralf R.; Campbell, Wilbur H.; Schwarz, Günter

doi:10.1105/tpc.104.029694

Cited by 149 publications

(137 citation statements)

References 51 publications

(72 reference statements)

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“…1 and Table I). The Mo-MPT and dimer interface domains of the nitrate-reducing module have recently been shown (29) to be closely associated as in mammalian and plant SOX (9, 30). Hinge 1 joins the Cyt b domain to the nitrate-reducing module.…”

Section: Resultsmentioning

confidence: 99%

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Barbier¹,

Campbell²

2005

Journal of Biological Chemistry

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Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD-(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.

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Section: Resultsmentioning

confidence: 99%

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Barbier¹,

Campbell²

2005

Journal of Biological Chemistry

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“…In addition, the pterin might also participate in the electron transfer to or from molybdenum via the delocalized electrons within the pterin (5). X-ray crystallographic analyses of molybdenum enzymes revealed that the cofactor is not located on the surface of the protein but is buried deeply within the interior of the enzyme, and a tunnel-like structure makes it accessible to the cognate substrates (12,13). During its lifetime, the molybdenum enzyme does not liberate Moco.…”

Section: What Is the Molybdenum Cofactor?mentioning

confidence: 99%

The Molybdenum Cofactor

Mendel

2013

Journal of Biological Chemistry

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The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Mocobinding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.The transition element molybdenum is an essential micronutrient for microorganisms, plants, and animals (1). Surprisingly, molybdenum itself is catalytically inactive in biological systems until it is complexed by a special scaffold (2). One type of scaffold is the ubiquitous pterin-based molybdenum cofactor (Moco), 2 which, in different variants, forms part of the active centers of all molybdenum enzymes in living organisms, except one. This exception is bacterial nitrogenase, which harbors the other type of cofactor, namely the Fe-S cluster-based FeMo cofactor, which is found only once in nature (for details, compare the minireview of Hu and Ribbe (62) in this thematic series). Molybdenum belongs to the group of trace elements, i.e. the organism needs it only in minute amounts; however, unavailability of molybdenum is lethal for the organism. Molybdenum is very abundant in the oceans in the form of the molybdate anion (3). In soils, the molybdate anion is the only form of molybdenum that is available for bacteria, plants, and fungi. More than 50 enzymes are known to be molybdenumdependent. The vast majority of them are found in bacteria, whereas only seven have been identified in eukaryotes (4, 5). It is somewhat surprising that not all organisms need molybdenum. The commonly used eukaryotic model organism yeast plays no role in molybdenum research, as Saccharomyces cerevisiae does not contain either molybdenum enzymes or the Moco biosynthesis pathway. Also Schizosaccharomyces pombe does not use molybdenum, whereas Pichia pastoris needs molybdenum. Genome-wide database analyses revealed a significant number of bacteria and unicellular eukaryotes that do not need molybdenum, whereas all multicellular eukaryotes are dependent on molybdenum (6). In addition, mainly anaerobic archaea and some bacteria are molybdenum-independent, but they require tungsten for their growth (7). In the periodic table of elements, tungsten lies directly below molybdenum and features chemical properties similar to molybdenum.Molybdenum metabolism is tightly connected to Fe-S cluster synthesis in that some of the molybdenum enzymes and Moco biosynthesis itself depend on Fe-S enzymes and on a mitochondrial transporter that is known to be crucial for the maturation of cytosolic Fe-S proteins. Moreover, Moco biosynthesis recruits mechanisms previously known from Fe-S cluster synthe...

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“…This causes the bond between this O atom and nitrate to break, forming nitrite which is subsequently released. Finally, transfer of two electrons and two protons from NAD(P)H via a cytochrome b5 domain cause regeneration of Mo 4 + and reduction of the O atom bound by a double bond to a water ligand [125].…”

Section: Ec 171: Oxidoreductases Acting On Other Nitrogenous Compoumentioning

confidence: 99%

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Vidal

Kelly

Mordaka

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

235

147

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A B S T R A C T NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P) + . NAD(P)Hdependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

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Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site

Cited by 149 publications

References 51 publications

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

The Molybdenum Cofactor

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

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