©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Nitrate Reductase from Aspergillus nidulans
IDENTIFICATION OF SOME ESSENTIAL AND SOME NONESSENTIAL AMINO ACIDS AMONG CONSERVED RESIDUES (*)

(Received for publication, December 12, 1994; and in revised form, January 24, 1995)

Julie Garde (1) James R. Kinghorn (2)(§) A. Brian Tomsett (1)(¶)

From the  (1)Department of Genetics and Microbiology, University of Liverpool, Liverpool, L69 3BX and the (2)Department of Biochemistry and Microbiology, University of St. Andrews, Fife, KY16 9AL, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. In Aspergillus nidulans, it is encoded by the niaD gene. A homologous transformation system has been used whereby a major deletion at the niiAniaD locus of the host was repaired by gene replacement. Employing site-directed mutagenesis and this transformation system, nine niaD mutants were generated carrying specific amino acid substitutions. Mutants in which alanine replaced cysteine 150, which is thought to bind the molybdenum atom of the molybdenum-pterin, and in which alanine replaced histidine 547, which putatively binds heme iron, had no detectable nitrate reductase (NAR) activity. This clearly establishes an essential catalytic role for these residues. Of the remaining mutants, all altered in the NADPH/FAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels. Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. These findings are considered in the light of predicted structural/functional roles for the altered amino acids.


INTRODUCTION

Inorganic nitrate can be used as a nitrogen source by fungi, algae, some yeasts, and some bacteria as well as by higher plants (see (1) and included reviews). These organisms perform nitrate assimilation as a sequential reduction of NO(3) to NO(2) and NO(2)to NH(4) by the action of two enzymes nitrate reductase (NAR) (^1)and nitrite reductase. Nitrate reductase is a complex multiredox enzyme possessing two identical subunits(2, 3) , each subunit containing three prosthetic groups held in separate domains. These comprise a molybdenum-containing pterin cofactor (MoCo), cytochrome b (heme) and FAD (see (4) and (5) ). The physiological activity of NAR involves a two-electron transfer from NAD(P)H, through the FAD, heme, and MoCo domains to NO(3), and this activity together with a number of nonphysiological partial activities of NAR can be assayed in vitro. The partial activities include the reduction of NO(3) by electrons passed to it from reduced methyl viologen (MVH) through Heme and MoCo, or bromphenol blue (BPB) through only MoCo(6, 7) .

A large number of fungal and plant NAR genes have been sequenced including niaD, the structural gene for Aspergillus nidulans NAR (see (8) and (9) ). From the deduced protein sequences, extensive homologies have been found between all eukaryotic NARs and between NAR and other redox enzymes. Human and bovine NADH cytochrome-b(5) reductase and spinach ferredoxin-NADP reductase show identity with the C-terminal FAD/NAD(P)H or cytochrome-b reductase domain. The small central Heme domain has 13 complete identities with all members of the cytochrome b(5) superfamily, and the N-terminal MoCo domain has homology with rat sulfite oxidase and, over limited regions, with Drosophila xanthine dehydrogenase(8, 9) . Between these catalytic domains lie less conserved hydrophilic hinge regions believed to be sites for cleavage by proteolytic enzymes, and shortened products of NAR derived from limited proteolysis and radiation inactivation have been shown to retain their partial activities indicating a functional independence for each of the catalytic domains(10, 11, 12) . This is further supported by the recent expression in Escherichia coli of catalytically active domains of corn and Chlorella NAR from partial cDNA clones (13, 14) .

In A. nidulans, the structural genes for NAR (niaD) and nitrite reductase (niiA) are divergently transcribed, and these two enzymes are co-regulated at the level of transcription(15, 16, 17) . They are subject to substrate induction by NO(3) or NO(2) mediated by the nirA gene product, and nitrogen metabolite repression mediated by ammonium/glutamine and the areA gene product.

Over several decades, many niaD mutants and mutants impaired in MoCo biosynthesis (cnx) have been characterized and shown to have multiple phenotypes. Many express the mutant NAR and nitrite reductase enzymes constitutively, i.e. even in the absence of inducer. This has led to the proposal that NAR has, in addition to its catalytic function, an autoregulatory function in preventing its own synthesis and that of nitrite reductase in the absence of inducer. The NAR-defective mutants also show varying degrees of chlorate resistance; constitutive synthesis correlates with chlorate resistance. Several are temperature-sensitive for NAR activity, and many retain one or more partial activities(18) . No molecular characterization of these mutants has yet been described, but a fine structure deletion map of niaD mutations has been published(16) .

To investigate, at the molecular level, the catalytic and putative regulatory function of the A. nidulans NAR enzyme, we have made nine new niaD mutant strains employing site-directed mutagenesis. To avoid anomalous regulatory responses, these were transformed into A. nidulans using a novel system (^2)that leads, in nearly every case, to homologous replacement of the niiAniaD gene locus without the integration of a second niaD gene and without remnants of defective niaD genes remaining in the genome. In these strains, specific amino acids have been changed that are implicated in NAR function on the basis of their conservation among all sequenced NARs and their homologous redox enzymes(9) , and/or on the basis of other studies which have implicated them to be involved in cofactor binding (MoCo(19) , Heme(20, 21, 22) , FAD(23) , or NADPH(24, 25) ) to the NAR apoenzyme. For the FAD/NADPH domain mutants, the catalytic activities are evaluated in the light of the structure of the cytochrome-b reductase domain of corn leaf NAR, which was determined subsequent to this study(26) .


MATERIALS AND METHODS

Site-directed Mutagenesis and Construction of Expression Vector

A restriction map of genomic DNA and the vectors are shown in Fig. 1. Genomic clones were constructed in pUC18, which lacked either the fragment (5` or N-terminal half) of the niaD gene (pUC/5`niiA/niaD5`DeltaB/K) or the beta fragment (3` or C-terminal half) of the niaD gene (pUC/5`niiA/niaD3`DeltaK/K). The genomic BamHI-SalI fragment (1802 bp = fragment + part of beta) that includes the N-terminal portion of the niaD gene and the overlapping KpnI-KpnI fragment (1961 bp = beta fragment) that includes the C-terminal portion were cloned, respectively, into M13mp18 and M13mp19 creating mp18 B/S and mp19 K/K. Uracil-containing single stranded DNA template was prepared from these M13 vectors using the dutungE. coli host CJ236, and site-directed mutagenesis was performed with mismatch primers (18-mer) and the appropriate template(27) . M13 plaques were screened by dideoxy sequencing (28) with Sequenase T(7) polymerase (U. S. Biochemical Corp.), and the nucleotide sequence of the insert from selected mutant clones was verified. The resulting BamHI-NotI fragment (1795 bp = fragment + part of beta) of mp18 B/S was cloned into pUC/5`niiA/niaD5`DeltaB/K (replacing a 446-bp BamHI-NotI fragment), and the KpnI-KpnI (beta) fragment of mp19 K/K was cloned into pUC/5`niiA/niaD3`DeltaK/K, in both cases creating pUC/5`niiA/niaD (see Fig. 1).


Figure 1: Vectors and their construction (not to scale). Restriction fragments and polymerase chain reaction-generated fragments were obtained from the original clones pSTA8, pILJ141, and pNIIA(17) . Restriction enzyme sites are as follows: X = Xho1; B = BamHI; K = KpnI; N = NotI; S = SalI; H = HindIII. The transcription of the two genes niiA and niaD are indicated by horizontalarrows above the shaded coding regions. The verticalarrows indicate the construction of pUC/5`niiA/niaD from the component parts (see text).



The recombinant phages and plasmids were constructed and characterized by standard procedures using the E. coli hosts JM105 and DH5alpha for the routine propagation of M13 bacteriophage and plasmids, respectively (see (29) ).

Protoplast Preparation and Transformation

Protoplasts of A. nidulans strain niiAniaDDelta503 (pabaA1) (16) were made by the method of Johnstone et al.(17) , except for final washing and resuspension in 1 ml of 10 mM CaCl(2), 50 mM Tris-HCl, pH 7.5, 1.2 M sorbitol. Plasmid DNA (10 µg, digested with DraI and NdeI to linearize the DNA), prepared according to Birnboim and Doly(30) , was added to 100 µl of protoplast suspension in 0.4-mm electroporation cuvettes and held on ice for 1 min. A Bio-Rad Gene Pulser with capacitance extender was used at the following settings: voltage 1700 V; resistance 200 ohms; capacitance 3 microfarads; capacitance extender 125 microfarads; time constant 0.6 ms. The suspension was immediately plated out onto selective medium in a molten overlay (2% agar over 1% agar). The selective medium was supplemented minimal medium (31) containing 1.2 M sorbitol and 10 mM potassium nitrite for the selection of niiA transformants.

Molecular and Phenotypic Characterization of A. nidulans Strains

Transformant homokaryons were purified by twice streaking single colonies onto nonsorbitol-containing selective medium. Total DNA was isolated from these homokaryon strains, untransformed host strains, and wild-type strain C5 (biA1) (31) by the method of Tilburn et al. (32). Conventional Southern blotting was performed (29) , and the blots were incubated in 10 ml of prehybridization solution (10% dextran sulfate, 1 mM NaCl, 1% SDS) for 4-5 h at 65 °C followed by the addition of denatured salmon sperm DNA (250 µg/ml) and P-labeled probe(33) , with continued incubation overnight.

To determine the phenotype of strains for growth on nitrate and chlorate toxicity, conidia were replicated from solid supplemented minimal medium containing potassium nitrite (10 mM) or ammonium tartrate (10 mM) as the nitrogen source onto the same medium containing potassium nitrate (10 mM) as the sole nitrogen source. Conidia were also replicated onto supplemented minimal medium containing the nitrogen sources arginine (10 mM), uric acid (10 mg/l), or proline (10 mM) with potassium chlorate added at concentrations of 0, 1, 10, or 100 mM. The plates were incubated at 37 °C (except for temperature sensitivity tests on nitrate at 30 °C) for 2-4 days before the phenotypes were scored.

Enzyme Assays

Aspergillus cultures were grown by shaking overnight in liquid-supplemented minimal medium at 37 or 30 °C with 10 mM urea or 10 mM urea plus 20 mM potassium nitrate as the nitrogen source. The mycelium was then harvested(31) , and cell-free extracts were produced by the method of MacDonald et al.(34) . The mycelium was extracted in fresh 0.1 M sodium phosphate buffer, pH 7.2, 5 mM EDTA, 5 mM cysteine, 10% glycerol for the nitrite reductase assay (nitrite reductase), and in 0.1 M sodium phosphate buffer, pH 6.8, 1 mM beta-mercaptoethanol, 0.5 mM EDTA, 1% NaCl for the nitrate reductase assay and its partial activity assays.

NADPH-nitrite reductase activity was measured as the oxidation of NADPH by a decrease in absorbance at 340 nm over 5 min as described by Pateman et al.(35) . NADPH-NAR activity was measured as the production of NO(2) by an increase in absorbance at 540 nm according to Garrett and Cove(36) . Reduced methyl viologen-NAR and bromphenol blue-NAR partial activities were measured in the same way and assayed by the procedures of Garrett and Nason (37) and Campbell(6) , respectively.

All of the enzyme activity assays were performed in triplicate and over two or more reaction times that gave a linear response. Protein concentrations were assayed by the Bradford method (38) using bovine serum albumen as the standard.


RESULTS

Production of Mutants

Two overlapping DNA fragments spanning the niaD coding region, fragment beta, and a fragment comprising + 443 bp of beta, were cloned separately into M13 (Fig. 1). Using the appropriate clone, nine base substitution mutants were made by site-directed mutagenesis. Table 1shows the DNA sequence, protein sequence, and approximate position of these mutations in relation to the three redox domains of the linear NAR protein.



niaDC150A occurs at a cysteine residue within the MoCo domain predicted to form a ligand with the molybdenum atom(19) . niaDH547A occurs at the putative first axial histidine of the heme domain presumed to bind to heme iron(20, 21, 22) . niaDW618K changes a residue conserved in fungi to one conserved in plants. niaDK626A is at a site conserved in all NAR molecules(9) . niaDH654A alters a residue implicated in NAD(P)H binding(24, 25) . niaDY693A alters a residue implicated in FAD binding(23) . niaDG721S was made to mimic a naturally isolated mutation so that the identity of the transgenic and natural mutations could be assessed. The natural mutant, niaD78, has a distinctive phenotype, being temperature sensitive and partially chlorate sensitive. niaDE850P and niaDEA850/1PP occur within the putative NADH/NADPH binding site at residues implicated in the determination of electron donor specificity(25) .

Mutant pUC/5`niiA/niaD plasmids were constructed by cloning either the KpnI-KpnI beta fragment into pUC/5`niiA/niaD3`DeltaK/K, or the BamHI-NotI extended fragment into pUC/5`niiA/niaD5`DeltaB/K (Fig. 1). Host strain niiAniaDDelta503 was transformed by electroporation to niiA with each mutant and wild-type plasmid (10 µg) using DraI-NdeI-cut DNA, and homokaryons of several transformants for each mutant were subjected to Southern blotting to confirm that homologous gene replacement had occurred (data not shown).

Phenotypic Characterization of Mutants: Growth Tests

By replica plating onto the appropriate agar medium, the mutant and wild type (TniaD) transformants, strain niaD78, and host strain niiAniaDDelta503, each were tested for their ability to grow on NO(3) at 37 and 30 °C and to grow in the presence of KClO(3) at 0, 10, and 100 µg/ml with arginine, uric acid, or proline as the nitrogen source. (ClO(3) toxicity is greatest with arginine and least with proline.) These results are summarized in Table 2. This analysis of the mutants supported the view that only the introduced mutation contributed to the phenotype. First, niaDG721S had essentially the same phenotype as strain niaD78, both grew on NO(3) at 30 °C but not 37 °C, and were ClO(3)-sensitive (the slightly greater KClO(3) sensitivity of niaDG721S is presumably due to its different genetic background). Second, all of the independent transformants of each mutant had identical phenotypes, and the wild types grew well on NO(3) and are ClO(3) sensitive as expected. There was no difference between the phenotype of transformants arising from homologous integration and replacement integration.



niaDC150A and niaDH547A, like the host strain, failed to grow on NO(3) and are completely chlorate resistant. niaDW618K is temperature sensitive for growth on NO(3) and has an intermediate ClO(3) sensitivity. niaDK626A, niaDH654A, niaDE850P, and niaDEA850/1PP all grow on NO(3) and are ClO(3) sensitive. niaDY693A also grows on NO(3) and is even more ClO(3) sensitive than the wild-type.

Phenotypic Characterization of Mutants: Enzyme Assays

Two independent transformants, for TniaD and each site-directed mutant, derived from replacement integration plus the wild-type C5 and niaD78 strains, were grown under inducing (10 mM urea + 20 mM NO(3)) and noninducing (10 mM urea) conditions and assayed for their nitrate reductase and nitrite reductase activities using crude mycelial extracts. In addition, niaDC150A and niaDH547A (which did not grow on NO(3) in the plate growth tests), niaDW618K and niaDG721S (which did not do so at 37 °C), niaDH654A (shown here to have very reduced nitrate reductase levels), and TniaD were also assayed for their BPB-NAR and MVH-NAR partial nitrate reductase activities. The partial BPB-NAR and MVH-NAR, and the NADPH-NAR activities measure, respectively, the function of the MoCo domain alone, the combined MoCo and Heme domains, and the combined MoCo, Heme, and FAD domains. The results of a typical experiment are presented in Table 3, showing the specific activities from each enzyme assay and the percentage values for the induced NAR and nitrite reductase levels relative to those for TniaD. The assays were performed after mycelial growth at 30 °C, which had been found to be optimum for NAR and nitrite reductase expression in TniaD and C(5) (Table 3) and also at 37 °C for TniaD and the temperature-sensitive niaDW618K, niaDG721S, and niaD78 (Table 3).



NAR and nitrite reductase expression for the identical mutants niaDG721S and niaD78 were very similar, as expected. These temperature-sensitive mutants had inducible NADPH-NAR activity at the permissive temperature (30 °C), although this was at a level much lower than in the wild-type strains, no such activity at the restrictive temperature (37 °C), and fully inducible nitrite reductase activity, comparable with wild-type at either temperature. niaDW618K, despite its temperature-sensitive phenotype, produced no detectable NADPH-NAR activity at 37 or 30 °C in the experiment shown, but low activity levels have been detected after growth at slightly lower temperatures and/or different culture conditions (data not shown); however, it had inducible nitrite reductase activity comparable with wild type. Mutants niaDW618K and niaDG721S also had inducible BPB-NAR and MVH-NAR activities after growth at 30 or 37 °C; whereas the induced BPB-NAR levels were comparable with wild-type, the induced MVH-NAR levels were manyfold higher, particularly for mutant niaDG721S. This was entirely consistent with a defect in the FAD domain for these mutants, the elevated MVH-NAR levels being due to partial denaturation of the NAR protein within the adjacent Heme domain leading to an increased accessibility for the electron donor methyl viologen. Greatly increased MVH-NAR levels have previously been demonstrated as a result of partial denaturation of the NAR protein (37) .

Mutants niaDC150A and niaDH547A had no NADPH-NAR activity, but did have constitutive NADPH-nitrite reductase activity at levels comparable with that of the induced wild-type. Mutant niaDH547A had no MVH-NAR activity but did have BPB-NAR activity expressed constitutively and at levels severalfold higher than that of the induced wild-type. This was entirely consistent with a defect in the heme domain, thus leading to increased accessibility, within the adjacent MoCo domain, for the electron donor BPB. niaDC150A has neither MVH-NAR or BPB-NAR activities consistent with a defect in the MoCo domain.

Mutants niaDK626A and niaDH654A showed a normal induction of NAR and nitrite reductase activities, but the induced NAR levels were considerably lower than for the wild-type, especially for mutant niaDH654A. This mutant also had inducible BPB-NAR activity comparable with the wild type and inducible MVH-NAR activity severalfold higher than the wild type, consistent with a defect in the FAD domain.

Mutants niaDE850P and niaDEA850/1PP showed a normal induction of NAR and nitrite reductase activities comparable with the wild type, as does niaDY693A, although NAR activity levels considerably higher than the wild type have been detected for this mutant after growth at 37 °C (data not shown).

Mutants niaDE850P, niaDEA850/1PP, and niaDH654A carry mutations at residues potentially involved in the binding of NADPH to NAR. NADPH has been shown to protect NAR against degradation in crude extracts of wild-type C5 incubated at raised temperatures(46) . In this study, such protection was demonstrated for the above mutants as well as for the wild-type TniaD and C5 strains, but no significant difference was found between the mutant and wild-type strains in the degree of protection afforded by NADPH (data not shown).

Mutants niaDE850Pand niaDEA850/1PP carry base substitutions that could potentially alter the affinity of NAR for NADPH and NADH. As described above, NADPH-NAR activities were comparable with the wild-type strains (TniaD and C5), but NADPH-NAR activity was not detected in either of these mutants or the wild-type strains (data not shown).


DISCUSSION

In this study, we have used a novel homologous transformation system in Aspergillus nidulans for structure/function studies of NAR by site-directed mutagenesis of the NAR structural gene, niaD. Using this system, positional or copy number effects did not occur, since the niaD gene integrated in single copy at the native locus. Mutant phenotypes could not be obscured due to recombination between transgenic and native niaD sequences or complementation between their encoded proteins, since the host lacks the whole niaD coding region. In confirmation of the expected accuracy of our system, between 3 and 9 transformants of the wild-type and each mutant were tested, and each produced identical phenotypes. A further test was the construction of niaDG721S, which was made identical to that of the classically isolated niaD78 mutation, and they had essentially the same phenotype. Analysis of key conserved residues throughout NAR could thus be undertaken with confidence.

In the MoCo domain, niaDC150A substituted alanine for Cys-150, the only totally conserved cysteine residue within NAR and sulfite oxidase(19) . The molobdenum-pterin cofactors that are common to these enzymes have 2-oxo and 2-3 thiol-like ligands coordinating the molybdenum atom(39) . It has been shown in MoCo-less nit1 mutant extracts of Neurospora crassa that a sulfydryl reagent blocks the reconstitution of NAR with exogenous MoCo, indicating that a thiolate-like ligand derives from the polypeptide, presumably the side chain of Cys-150(40) . Consistent with this prediction, niaDC150A totally abolishes NAR activity as well as the BPB-NAR and MVH-NAR partial activities. There is evidence in plants for a disulfide bond involved in NAR dimerization(41) . Whether the niaDC150A NAR protein is MoCo-less or monomeric are important questions still to be addressed.

niaDH547A substitutes alanine for His-547, the putative first axial histidine of the heme domain. Two histidines out of 11 residues totally conserved in every NAR and cytochrome b(5) so far analyzed, were shown to bind heme iron in bovine cytochrome b(5) upon elucidation of the crystal structure(22) . A very similar structure is predicted for the NAR heme domain(21) . In a natural NAR mutant of N. plumbaginifolia the putative first iron binding histidine was shown to be replaced by asparagine, but some substitution of function was indicated as heme still bound to the mutant NAR, although producing an abnormal spectral scan, and a very low level of two heme-requiring partial activities were detected(21) . In mutant niaDH547A, we found absolutely no NAR activity or heme-requiring MVH-NAR activity, but we found a high level of MoCo-requiring BPB-NAR activity. This is consistent with the putative role of His-547 in heme iron binding and the abolition of this function by substitution with alanine. However, exactly the same mutation made by site-directed mutagenesis in Neurospora did not greatly reduce NAR activity(42) .

For the analysis of the FAD (cytochrome-b reductase) domain, seven mutations were made based upon amino acid conservation/differences in various NAR molecules. Subsequent to this study, a structure has been determined for the corn leaf NADH-NAR recombinant cytochrome-b reductase domain bound with FAD, and this throws further light upon the catalytic activities found for the FAD/NADPH-binding domain mutants. The corn leaf NAR cytochrome-b reductase domain comprises two lobes separated by a short linker(26) . The N-terminal FAD-binding lobe consists of a six antiparallel stranded beta-barrels with one alpha-helix. The C-terminal NADH-binding lobe has a six parallel stranded beta-sheet flanked by two alpha helices. This a variant of the classical dinucleotide binding fold. Superimposing the A. nidulans amino acid sequence upon this structure by homologous replacement, further observations can be made.

niaDY693A substitutes alanine for Tyr-693, one of only two totally conserved tyrosines in NAR and cytochrome-b(5) reductase(9) . Group-specific modification of cytochrome-b(5) reductase has shown that a single tyrosine participates in FAD binding(23) . We have ruled out Tyr-693 as a candidate for this activity as niaDY693A did not inhibit NAR activity; instead, after growth at 37 °C, this activity was slightly increased. From the structure of the N-terminal lobe(26) , it can be seen that while the adenine ribose moiety of FAD is in close proximity to Tyr-693, it does not form any bonds with the protein. Furthermore, in ferredoxin NADP reductase, this moiety adopts a different conformation suggesting that its orientation is not important for biological function(26) . This explains the absence of catalytic effect for niaDY693A. On the other hand, the only other conserved tyrosine of the FAD domain bonds through its hydroxyl group to the O(4) atom in the ribityl moiety(26) .

niaDG721S substituted serine for glycine 721, which is totally conserved in all NARs and cytochrome-b(5) reductase(9) . This change confers temperature sensitivity for growth on nitrate and inducible MVH-NAR and BPB-NAR partial activities at the restrictive temperature, indicating a potential catalytic role for Gly-721 within the FAD domain. Gly-721 lies within the beta-barrel, and thus the substitution in niaDG721S may have structural consequences for the binding of FAD, resulting in the altered activity.

niaDH654A substitutes alanine for histidine 654, the only totally conserved histidine in NAR and cytochrome-b(5) reductase. A report of an inhibition of dehydrogenase activity for Amaranthus NAR by histidine-specific reagents and protection by NADH suggests His-654 could be at the active site involved in binding pyridine dinucleotide (24) . However, niaDH654A was found to retain about 30% of the wild-type NAR activity and to possess MVH-NAR and BPB-NAR activities similar to or above that of the wild-type, indicating an important role for His-654 in the FAD domain, although one that is not essential for catalysis but merely facilitates it. His-654 lies at the start of the third beta-strand, and thus the consequences of the niaDH654A mutation perhaps arise from its position within a cleft through which the heme of the cytochrome b may gain access to the FAD.

niaDK626A changes lysine 626 to alanine, a residue that might be assumed to have some structural/functional importance on the basis that it is conserved in all NARs. It possessed inducible NAR activity to about 55% of the wild-type level, indicating a facilitating catalytic role for Lys-626, which lies within the first beta-strand of the FAD-binding lobe. This mutant was also inducible for nitrite reductase activity expressed at normal wild-type levels, which rules out any autoregulation function for Lys-626.

niaDW618K changes tryptophan-618, which is conserved in fungal NARs, to lysine, which is conserved at this position in all plant NARs, thus altering a candidate residue for involvement in the putative fungal specific autoregulatory activity of NAR. niaDW618K was found to be temperature-sensitive for NAR activity, being very weakly active at or just below 30 °C, and to possess inducible BPB-NAR and MVH-NAR partial activities at the restrictive temperature, consistent with a catalytic role for Trp-618 in the FAD domain, perhaps in folding to enable FAD to orientate correctly in relation to the heme and MoCo centers. Trp-618 occurs at the start of the first beta-strand in the FAD-binding lobe, some considerable distance from the FAD-binding site, but it is close to the position of a cysteine residue of corn NAR, which although it is not conserved, has been shown to be functionally important(26) . Mutations around this region, including niaDW618K, might in general introduce structural change that affect distant sites. The fact that this mutant retained normal inducibility for nitrite reductase activity rules out any autoregulation function.

niaDE850P and niaDEA850/1PP alter residues in the C-terminal NADPH binding lobe of the cytochrome-b reductase domain. There is a conserved motif that has the sequence CGPEAM in fungal NAR, CGPPPM in most plant NADH-NARs and cytochrome-b(5) reductase, and CGAPSM in the birch NAD(P)H bispecific NAR(25) . The motif was thought to define the pyridine nucleotide binding site as an essential catalytic thiol for NADH binding in Chlorella NAR, and cytochrome-b(5) reductase has been localized to this region(43, 44) . Very recently, an essential catalytic function for the cysteine of this motif has been found by mutational analysis of the FAD domain of corn leaf NAR expressed in E. coli(45) . Friemann et al.(25) have proposed that the residues within this motif that differ between fungal NADPH-NAR, the birch NAD(P)H-NAR, and plant NADH-NAR might specify the choice of electron donor. niaDE850P replaces glutamic acid 850 with proline, and niaDEA850/1PP replaces glutamic acid 850 with proline and alanine 851 with proline, thus creating sequences more similar to the bispecific NAR and the plant NADH-NARs, respectively. Neither of these mutations had any effect upon NADPH-NAR activity nor did they confer any NADH-NAR activity or alter the ability of NADPH to protect NAR from degradation at high temperature, a feature previously reported(46) . We conclude that E-850/P and EA-850/1PP have no primary role upon the choice of electron donor for NAR; instead, if they have any role in higher plant NARs, it must be secondary, for example to optimize conformation due to differences in other nonconserved regions of this domain.

It can be seen from the structure that Glu-850 and Ala-851 lie on a very short alpha-helix that packs against the ADP moiety of the pyridine nucleotide(26) . Specificity for NADH in the corn leaf enzyme may be at least partly conferred by a negatively charged residue on the third beta-strand (Asp-205, equivalent to Thr-813 in A. nidulans), which binds the 2`OH group of NADH and causes the repulsion of the 2` phosphate group of NADPH. This awaits experimental confirmation.

It can be seen from these results that the use of our homologous transformation system has proven to be a simple and accurate way of expressing the transgenic niaD gene and has allowed the powerful tool of site-directed mutagenesis to be used to directly test the functional role of candidate residues in whole NAR apoprotein in vivo. We have established a critical catalytic role for the totally conserved residues His-547, the putative first axial histidine involved in heme binding, and Cys-150, which may bind the molybdenum atom, and could participate in subunit dimerization. We have found no direct role for Glu-850 or Glu-850 + Ala-851 in specifying the use of NADPH, as the electron donor for fungal NAR. A catalytic role for Trp-618, conserved only in fungal NAR, and for the totally conserved residue Gly-721, have been found. Tyr-693 appears to have no direct role, and His-654 and Lys-626 have only a facilitating role. This is perhaps surprising since these latter three residues are totally conserved in NAR, and in the case of Tyr-693 and His-654 also cytochrome-b(5) reductase. These findings not only challenge the predicted role of Glu-850 and EA-850/1 in the determination of electron donor specificity, Tyr-693 in FAD binding, and His-654 in NADPH binding, they challenge any assumption that the degree of conservation of individual amino acids always correlates with their structural/functional status. We anticipate that our approach to the study of the structure/function relationships of NAR will become especially important with the future elucidation of the structure of NAR, as has already been accomplished for the corn leaf cytochrome-b reductase domain (26) and with continuing biophysical studies on the catalytic domains of this enzyme.


FOOTNOTES

*
This research was supported by Grant GR/F97089 from the Science and Engineering Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Plant Science Laboratory, Sir Harold Mitchell Bldg., School of Biological and Medical Sciences, University of St. Andrews, Fife KY196 9AL, UK.

To whom correspondence should be addressed: Dept. of Genetics and Microbiology, The University of Liverpool, Donnan Laboratories, P. O. Box 147, Liverpool L69 3BX, UK. Tel.: 44-51-794-3616; Fax: 44-51-794-3655; tomsett{at}liverpool.ac.uk.

(^1)
The abbreviations used are: NAR, nitrate reductase; MoCo, molybdenum-pterin cofactor; bp, base pairs; MVH, reduced methyl viologen; BPB, bromphenol blue; WT, wild type; PCR, polymerase chain reaction.

(^2)
J. Garde, J. R. Kinghorn, and A. B. Tomsett, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Lesley Andrews for technical assistance. We also thank Wilbur Campbell and Neil Hall for helpful discussions and critical reading of the manuscript.


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