High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase

Hyde, Gregory E.; Campbell, Wilbur H.

doi:10.1016/0006-291x(90)91168-r

Cited by 48 publications

(29 citation statements)

References 19 publications

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“…Cyt c reductase activity was eluted from the Zm2(69) Sepharose in a sharp peak, which absorbed light at 413 nm, a characteristic ofthe heme-Fe cofactor ofNR (Table I). The specific activity of the Cyt c reductase activity was increased 800-fold in the peak fraction (fraction 7) of the Zm2(69) Sepharose pH 11 elution, in which 60% of the activity bound to the column was recovered (Table I). When NADPH was substituted for NADH in the Cyt c reductase assay, the purified recombinant enzyme was slightly more active than maize leaf NADH:NR with this substrate but clearly had a strong preference for NADH (Table II).…”

Section: Purification Of Recombinant Cyt C Reductasementioning

confidence: 96%

“…Although the recombinant Cyt c reductase was not expressed at a high level, ase (CCR) and maize the system will be useful for determining essential amino acid pH 11 elution from residues of the Cyt b and FAD domains of NR via sited ----) spectra were directed mutagenesis, especially when studied in combination J Methods." Proteins with the recombinant FAD domain which is expressed at a high level ( 11).…”

Section: Reductasementioning

confidence: 99%

“…The FAD domain of maize leaf NADH:NR was overexpressed in E. coli using part of the Zmnrl cDNA (9) and purified to homogeneity by NADH elution from blue Sepharose (11). The recombinant FAD domain of NR has an Mr of 30,000, NADH:ferricyanide reductase activity, and a UV-visible spectrum essentially identical with mammalian NADH:Cyt b5 reductase, which demonstrated that FAD is bound to the recombinant protein in a functional manner (1 1).…”

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confidence: 99%

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Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

Campbell¹

1992

Plant Physiol.

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A cDNA clone was isolated from a maize (Zea mays L. cv W64AxW183E) scutellum Xgtl 1 library using maize leaf NADH:nitrate reductase Zmnrl cDNA clone as a hybridization probe; it was designated Zmnr1S. ZmnrlS was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnrl. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.Higher plant NADH:NR2 (EC 1.6.6.1) catalyzes the first step in nitrate assimilation and is a key site for regulation of this pathway (22). The enzyme is a homodimer composed of two approximately 100-kD polypeptide chains with each containing one equivalent of FAD, heme-Fe, and 3,17). Each of these electron-carrying cofactors appears to be bound into a specific region of the enzyme's polypeptide, which folds into distinct domains. The N-terminal region of NR can be recognized as the Mo-pterin domain by comparing

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Section: Purification Of Recombinant Cyt C Reductasementioning

confidence: 96%

Section: Reductasementioning

confidence: 99%

mentioning

confidence: 99%

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Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

Campbell¹

1992

Plant Physiol.

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“…For some time, there has been a need for a system to express plant NR cDNAs for both structural and regulatory studies. Fragments of NR have been produced in bacteria and yeast (Hyde and Campbell, 1990;Campbell, 1992;Cannons et al, 1993;Cannons and Solomonson, 1994;Shiraishi and Campbell, 1995), and an apo-NR has been made in S. cerevisiae (Truong et al, 1991). It is not surprising that the yeast S. cerevisiae cannot make holo-NR because this yeast cannot In some systems, aspartate has been shown to mimic a phosphorylated serine.…”

Section: Discussionmentioning

confidence: 99%

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Huber

Crawford

1996

Plant Cell

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Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in COz levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIAí and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. lncubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in F! pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach.A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect.The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plant NRs, as an essential site for post-translational regulation in vitro.

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“…All mid-point redox potentials were converted to the standard hydrogen electrode potential. ZmCbR was prepared as previously described (Hyde and Campbell, 1990;Campbell, 1992).…”

Section: Electrochemical Analysismentioning

confidence: 99%

Recombinant Expression of Molybdenum Reductase Fragments of Plant Nitrate Reductase at High Levels in Pichia pastoris

2000

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Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO3 reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L−1, corn MoR at 32 mg L−1, and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L−1. Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.

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High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase

Cited by 48 publications

References 19 publications

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Recombinant Expression of Molybdenum Reductase Fragments of Plant Nitrate Reductase at High Levels in Pichia pastoris

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