Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

Barbier, Guillaume G.; Joshi, Rama C; Campbell, Ellen R.; Campbell, Wilbur H.

doi:10.1016/j.pep.2004.05.021

Cited by 33 publications

(32 citation statements)

References 19 publications

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“…7 However, it has also been reported that some other yeasts, such as Candida nitratophila, Pichia anomala, and Pichia angusta, utilize Mo-containing assimilatory NR. [42][43][44] In the present study, we could not detect homologs of such NR in sequenced yeast genomes, including Candida albicans, Candida glabrata, Candida tropicalis, and Pichia guilliermondii. The absence of both Moco biosynthesis pathway and assimilatory NR strongly suggested the loss of Mo utilization in most yeast species.…”

Section: Occurrence Of Molybdoenzymes In Prokaryotes and Eukaryotescontrasting

confidence: 70%

Molybdoproteomes and Evolution of Molybdenum Utilization

Zhang

Gladyshev

2008

Journal of Molecular Biology

179

206

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Section: Occurrence Of Molybdoenzymes In Prokaryotes and Eukaryotescontrasting

confidence: 70%

Molybdoproteomes and Evolution of Molybdenum Utilization

Zhang

Gladyshev

2008

Journal of Molecular Biology

179

206

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“…Approximayely 1 mg of linearized plasmid was used to transform electrocompetent wild-type P. pastoris by electroporation. After NR-Mo expressing cell line selection, fermentation was performed as described for the related fragment of YNR1, which is called simplified NR or S-NaR1 (Barbier et al, 2004). The disruption of the cells and the purification of the crude NR-Mo by immobilized metal affinity chromatography were done according to the protocol previously described for S-NaR1.…”

Section: Methods Protein Expression and Purificationmentioning

confidence: 99%

“…Analysis of a fragment containing heme and FAD domains of spinach (Spinacia oleracea) NR indicated that electron transfer from heme appeared to be slightly slower than FAD reduction (Ratnam et al, 1997). For the spinach NR Mo domain, nitrate K m is 8 to 12 mM (Pollock et al, 2002); whereas for the corresponding nitrate-reducing fragment from yeast NR, the nitrate K m is 30 mM (Barbier et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 Å. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis

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“…Yeast NaR (YNaR1) was obtained by cloning the YNaR1 gene from Pichia angusta (24) with N-terminal His tag into an appropriate expression vector and production in Pichia pastoris (results not shown). YNaR1 was purified by metal-chelate affinity chromatography, as described for S-NaR1, its recombinant nitrate-reducing fragment (25). The methods used to obtain Z. mays Cyt b reductase fragment (ZmCbR), spinach molybdenum reductase (SoMoR), and corn molybdenum reductase (ZmMoR) were previously described (12, 26 -28).…”

Section: Methodsmentioning

confidence: 99%

“…Each experiment was done twice, and representative data are shown. MV:NaR and Cyt c reductase activity assays were done with YNaR1 as previously described (12,25) at 0 and 50% glycerol.…”

Section: Methodsmentioning

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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Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

Cited by 33 publications

References 19 publications

Molybdoproteomes and Evolution of Molybdenum Utilization

Molybdoproteomes and Evolution of Molybdenum Utilization

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

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

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