(Received for publication, December 12, 1994; and in revised form, January 24, 1995)
From the
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.
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 to NO
and
NO
to NH
by
the action of two enzymes nitrate reductase (NAR) (
)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
, 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
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 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
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 or
NO
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 ()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) .
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 DH5 for the routine
propagation of M13 bacteriophage and plasmids, respectively (see (29) ).
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.
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 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.
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 fragment into
pUC/5`niiA/niaD3`
K/K, or the BamHI-NotI
extended
fragment into pUC/5`niiA/niaD5`
B/K (Fig. 1). Host strain niiAniaD
503 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).
niaDC150A and niaDH547A, like the host strain, failed to grow on
NO and are completely chlorate resistant. niaDW618K is temperature sensitive for growth on
NO
and has an intermediate
ClO
sensitivity. niaDK626A,
niaDH654A, niaDE850P, and niaDEA850/1PP all grow on
NO
and are ClO
sensitive. niaDY693A also grows on
NO
and is even more
ClO
sensitive than the wild-type.
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).
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 so far analyzed, were shown to bind heme iron in
bovine cytochrome b
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 -barrels with one
-helix. The
C-terminal NADH-binding lobe has a six parallel stranded
-sheet
flanked by two
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 reductase(9) . Group-specific
modification of cytochrome-b
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
atom in the ribityl moiety(26) .
niaDG721S substituted serine for glycine 721, which is
totally conserved in all NARs and cytochrome-b 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
-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 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
-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 -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 -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 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
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 -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
-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 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.