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W H Campbell

Publications and source records attributed to W H Campbell.

At least 19 recordsLinked to original sources
PubMed2001

Pre-steady-state kinetic analysis of recombinant Arabidopsis NADH:nitrate reductase: rate-limiting processes in catalysis.

Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation. Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase. Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin. Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.

Arabidopsis↗
PubMed2001

Structure and function of eukaryotic NAD(P)H:nitrate reductase.

Pyridine nucleotide-dependent nitrate reductases (NRs; EC 1.6.6.1-3) are molybdenum-containing enzymes found in eukaryotic organisms which assimilate nitrate. NR is a homodimer with an approximately 100 kDa polypeptide which folds into stable domains housing each of the enzyme's redox cofactors--FAD, heme-Fe molybdopterin (Mo-MPT) and the electron donor NAD(P)H--and there is also a domain for the dimer interface. NR has two active sites: the nitrate-reducing Mo-containing active site and the pyridine nucleotide active site formed between the FAD and NAD(P)H domains. The major barriers to defining the mechanism of catalysis for NR are obtaining the detailed three-dimensional structures for oxidized and reduced enzyme and more in-depth analysis of electron transfer rates in holo-NR. Recombinant expression of holo-NR and its fragments, including site-directed mutagenesis of key acative site and domain interface residues, are expected to make large contributions to this effort to understand the catalytic mechanism of NR.

Catalytic Domain↗
PubMed2000

Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris.

Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) 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.

Aldehyde Oxidoreductases↗
PubMed1998

Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development.

Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68). Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season. These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season. To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid. Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates. The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms. We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution. Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively. This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms.

Base Sequence↗
PubMed1998

Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase.

The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).

Amino Acid Sequence↗
PubMed1998

Construction and characterization of nitrate reductase-based amperometric electrode and nitrate assay of fertilizers and drinking water.

The construction and characterization of a nitrate reductase-based amperometric electrode for determination of nitrate ion is described. The electrode consisted of nitrate reductase held by dialysis membrane onto a Nafion-coated glassy carbon electrode. Methyl viologen was allowed to absorb into the Nafion layer, which acted as a reservoir for the electron mediator. The utility of the electrode to assay fertilizer and water sample for nitrate was demonstrated. The assays conducted with this electrode compared well with colorimetric and potentiometric assays of the same samples.

Absorption↗
PubMed1997

Spectroscopic and kinetic characterization of the recombinant cytochrome c reductase fragment of nitrate reductase. Identification of the rate-limiting catalytic step.

The recombinant NADH-cytochrome c reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1), consisting of the contiguous heme-containing cytochrome b domain and flavin-containing NADH-cytochrome b reductase fragment, has been characterized spectroscopically and kinetically. Reductive titration with sodium dithionite indicates heme reduction takes place prior to flavin reduction, which correlates well with the reduction potentials for enzyme-bound heme (15 mV) and FAD (-280 mV). Reductive titration with NADH also indicates that the reduced enzyme forms a charge-transfer complex with NAD+. The circular dichroism spectrum of the oxidized fragment is primarily due to the flavin, whereas the ferrous heme dominates the circular dichroism spectrum of reduced enzyme. Three kinetic phases are observed in the course of the reaction of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the formation of a charge-transfer complex between reduced flavin and NAD+, and exhibits hyperbolic dependence on NADH concentration with a Kd of 3 microM and a limiting rate constant of 560 s-1. Electron transfer from reduced flavin to heme with a rate constant of 12 s-1 is the intermediate phase, which is rate-limited by breakdown of the charge-transfer complex between NAD+ and reduced flavin. The slow phase is dismutation of a pair of molecules of two-electron reduced enzyme (generated at the end of the second phase of the reaction) to give one molecule each of one- and three- electron reduced enzyme, with a second order rate constant of 2 x 10(6) M-1 s-1. In the presence of excess NADH, this dismutation reaction is followed by the rapid reaction of the one-electron reduced enzyme with a second equivalent of NADH to generate fully reduced enzyme. On the basis of this work, it appears that dissociation of NAD+ from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limiting step of catalysis.

Binding Sites↗
PubMed1997

Misplaced rewards: Veterans' Administration system and symptom magnification.

The process of disability determination in the Veterans' Administration system and the maladaptive patterns of behavior that may develop in veterans attempting to qualify for disability benefits have been subjects of considerable interest to those who care for these individuals. The potential misuse of disability payments by veterans, especially for purposes of financing drug and alcohol dependence, is a related issue of concern. The quest for disability status is frequently an all consuming process, as the veteran attempts to document the presence and severity of his or her disorder to the degree necessary to obtain approval for compensation. Veterans' Administration patients may engage in symptom magnification as a means of convincing healthcare workers of the veracity of their complaints and to establish the necessary paper trail for their disability claims. It is clear when dealing with this challenging patient population that it is exceedingly difficult to know when a patient has a prominent psychologic overlay to his or her condition, is embellishing existing symptoms, or is prevaricating in an effort to obtain disability payments. Although it is essential to focus on the objective signs of disease while attempting to evaluate the subjective correlates, it is also important to determine the motivation of the patient. Rewarding individuals for pain and disability, particularly on a long term basis, can have numerous negative consequences and ultimately may be a disservice to the patient.

Adult↗
PubMed1997

Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris.

Recombinant Arabidopsis thaliana NADH:nitrate reductase (NR; EC 1.6.6.1) was produced in the methylotrophic yeast Pichia pastoris and purified to near-electrophoretic homogeneity. Purified enzyme had the spectral and kinetic properties typical of highly purified NR from natural plant sources. Site-directed mutagenesis altering several key residues and regions was carried out, and the mutant enzyme forms were expressed in P. pastoris. When the invariant cysteine residue, cysteine-191, in the molybdo-pterin region of the A. thaliana NIA2 protein was replaced with serine or alanine, the NR protein was still produced but was inactive, showing that this residue is essential for enzyme activity. Deletions or substitutions of the conserved N terminus of NR retained activity and the ability to be inactivated in vitro when incubated with ATP. Enzyme with a histidine sequence appended to the N terminus was still active and was easily purified using metal-chelate affinity chromatography. These results demonstrate that P. pastoris is a useful and reliable system for producing recombinant holo-NR from plants.

Arabidopsis↗
PubMed1996

Characterization and site-directed mutagenesis of aspen lignin-specific O-methyltransferase expressed in Escherichia coli.

Aspen lignin-specific caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (EC 1.2.1.68) was expressed in an active form in Escherichia coli using pET-23 vector. Two steps were used to purify (Phenyl Sepharose and S-adenosylhomocysteine-agarose chromatographies) enzyme to homogeneity. O-Methyl-transferase has a subunit of 40 kDa and native gradient gel electrophoresis indicated the active form is a dimer. Substrate specificity was investigated using over 20 phenolic compounds, which defined the nature of the substrate binding site and required substrate characteristics such as a hydroxyl group para to the side chain. Enzyme accommodates large substrates well if the side chain contains the trans-double bond found in lignin precursors. Kinetically S-adenosyl-L-methionine must bind before phenolic substrate; however, S-adenosyl-L-homocysteine and phenolic substrate or product can form stable complexes complicating the kinetic mechanism. The role of thiol side chain(s) in the catalytic mechanism was investigated since the enzyme is inhibited by p-chloromercuribenzoate. Of nine cysteine residues in the enzyme's sequence, only cysteine residues at positions 276 and 283 are invariant among higher plant O-methyltransferases of this class. These residues were replaced by serine and alanine, singly and in combination, using site-directed mutagenesis. All combinations of cysteine replacements at positions 276 and 283 yielded enzyme virtually as active as wild-type and all were still sensitive to thiol inhibition. We concluded that thiol(s) were not important in the catalytic mechanism of this class of O-methyltransferases and sensitivity to the large thiol inhibitor was probably due to reaction of cysteine thiol(s) near the surface which sterically hindered the active site.

Amino Acid Sequence↗
PubMed1996

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

Amino Acid Sequence↗
PubMed1995

Spectroscopic and kinetic characterization of the recombinant wild-type and C242S mutant of the cytochrome b reductase fragment of nitrate reductase.

Spectroscopic and kinetic studies comparing the behavior of the recombinant cytochrome b reductase fragment of corn leaf nitrate reductase and a mutant in which cysteine 242 is replaced with a serine residue (C242S) have been carried out. The visible and circular dichroism spectra of the wild-type and mutant protein are virtually identical and compare well with those reported for nitrate reductases from other sources. The reduced wild-type protein forms a charge-transfer complex with NAD+ that has an absorption envelope that extends into the near infrared, with a maximum around 800 nm. The C242S mutant forms a similar charge-transfer complex with NAD+ but to a lesser extent than the wild-type. The reduction potential of the flavin for the wild-type protein is -287 mV, and that for the mutant is -279 mV. The rate of reduction by NADH of the C242S mutant is 7-fold slower than that for the wild-type protein, and the Kd is larger by a factor of 2. These results indicate that the cysteine 242 residue plays a role principally in facilitating electron transfer from NADH to the flavin rather than in binding of NADH to the enzyme.

Circular Dichroism↗
PubMed1994

Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5 A resolution: relationship to other flavoprotein reductases.

BACKGROUND: In the biological assimilation of nitrate in plants and microorganisms, nitrate is reduced to ammonium by transfer of eight electrons in a two-step process. The first step of the pathway, the reduction of nitrate to nitrite, is catalyzed by nitrate reductase, a multi-redox cofactor enzyme which belongs to the class of flavoprotein pyridine nucleotide cytochrome reductases. The enzyme can be divided into three functional fragments that bind the cofactors molybdopterin, heme-iron and flavin adenine dinucleotide (FAD)/nicotinamide adenine dinucleotide (NADH). RESULTS: Here we describe the crystal structure of the recombinant cytochrome b reductase fragment of corn nitrate reductase, in complex with the cofactor FAD, determined to 2.5 A resolution. This catalytically competent fragment of nitrate reductase consists of two domains, the amino-terminal lobe, which binds FAD, and the carboxy-terminal lobe, which presumably binds NADH, connected by a linker region. CONCLUSIONS: Nitrate reductase belongs to the class of flavoprotein pyridine nucleotide cytochrome reductases, a subgroup in the family of ferredoxin reductase-like flavoproteins. Comparison with other members of this family reveals that large structural differences are found in the relative orientation of the cofactor binding lobes. This indicates that conformational changes might be important for biological function.

Amino Acid Sequence↗
PubMed1994

Identification of an "essential" cysteine of nitrate reductase via mutagenesis of its recombinant cytochrome b reductase domain.

Five cysteine residues in the recombinant cytochrome b reductase domain of corn leaf NADH:nitrate reductase (EC 1.6.6.1) were modified by site-directed mutagenesis. At least two amino acid replacement mutants were generated for each of the 5 cysteines of this domain. Characteristics of the amino acid replacement mutants correlated well with the structural location of the cysteine residues in the preliminary three-dimensional model of the cytochrome b reductase domain: somewhat exposed cysteines could be replaced by hydrophilic amino acid residues, while more buried cysteines by hydrophobic residues. An exception was found for the invariant cysteine near the C terminus, which is found in all nitrate reductases and also in the closely related NADH: cytochrome b5 reductase, as well as, most other members of this flavoenzyme family. No substitution for the invariant cysteine yielded highly active enzyme, although these mutants had normal visible spectra. When the invariant cysteine was mutated to serine, the cytochrome b reductase domain was resistant to inhibition by pchloromercuribenzoate, an inhibitor of nitrate reductases. Kinetic analysis suggested that the catalytic efficiency of the mutant was markedly reduced. We concluded, the invariant cysteine plays an important role in catalysis and may be essential for high catalytic efficiency of nitrate reductases.

Amino Acid Sequence↗
PubMed1994

Identification of a maize root transcript expressed in the primary response to nitrate: characterization of a cDNA with homology to ferredoxin-NADP+ oxidoreductase.

To more fully understand the biochemical and molecular events which occur in plants exposed to nitrate, cDNAs whose accumulation was enhanced in nitrate- and cycloheximide-treated maize (Zea mays L. W64A x W182E) roots were isolated. The 340 bp Zmrprn1 (for Zea mays root primary response to nitrate) cDNA also hybridized with a probe enriched for nitrate-induced sequences, and was characterized further. Sequence analysis of a near full-length cDNA (Zmrprn1A) showed strong homology (> 90% amino acid identity) with a root ferredoxin-NADP+ oxidoreductase (FNR) of rice, and 45-50% amino acid identify with leaf FNR genes. When expressed in Escherichia coli, the Zmrprn1A cDNA produced a protein with NADPH: ferricyanide reductase activity, consistent with the enzymatic properties of an FNR. The Zmrprn1 cDNA hybridized with a 1.4 kb transcript which was expressed in the maize root primary response to nitrate. That is, mRNA levels in roots increased rapidly and transiently in response to external nitrate, and low levels of nitrate (10 microM) induced transcript accumulation. The accumulation of the Zmrprn1 transcript was not prevented by cycloheximide, indicating that the cellular factor(s) required for expression were constitutively present in maize roots. The Zmrprn1 mRNA accumulated specifically in response to nitrate, since neither K+ nor NH4+ treatment of roots caused transcript accumulation. Maize leaves had about 5% of the transcript level found in roots, indicating a strong preference for expression of Zmrprn1 in roots. Analysis of maize genomic DNA indicated the presence of only a single gene or very small gene family for the Zmrprn1. Together, the data indicate that Zmrprn1A encodes a nitrate regulated maize root FNR.

Amino Acid Sequence↗
PubMed1994

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus.

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-L-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.

Base Sequence↗