Characterization of pco-1, a newly identified gene which regulates purine catabolism in Neurospora.

A new gene of Neurospora crassa, designated pco-1, was characterized and shown to regulate the expression of several genes which encode enzymes required for the catabolism of purines. Unlike the wild type, a pco-1 mutant created by repeat-induced point mutation cannot utilize purines as a nitrogen source. The PCO1 protein contains a Zn(II)2Cys6 binuclear cluster motif near its N-terminus, followed by a putative coiled-coil motif. A chemical crosslinking experiment demonstrated that PCO1 forms homodimers. PCO1 binds to CGG-N6-CCG elements located in the upstream promoter region of four genes encoding purine catabolic enzymes. Northern blot analysis demonstrated that a functional PCO1 protein is required for induction of xdh, which encodes xanthine dehydrogenase. Moreover, PCO1 was required for induction of three different purine catabolic enzymes. Two glutamine-rich domains occur in the C-terminal region of PCO1 and at least one of the glutamine-rich regions is required for PCO1 function, suggesting that they might play a role in transcriptional activation. The PCO1 protein does not interact with the global-acting NIT2 protein or the negative-acting NMR protein that functions in nitrogen catabolite repression. Induction of the xdh gene and synthesis of xanthine dehydrogenase is completely dependent upon PCO1, but does not require the global-acting NIT2 protein, suggesting that it is controlled by a novel regulatory mechanism.

ASD4, a new GATA factor of Neurospora crassa, displays sequence-specific DNA binding and functions in ascus and ascospore development.

A new gene encoding a novel GATA factor, ASD4, of Neurospora crassa was isolated and demonstrated to possess one intron and to specify an open reading frame encoding a protein with 427 amino acid residues. The ASD4 protein contains a single GATA-type zinc finger and a putative coiled-coil domain. Unlike related proteins, DAL80 in yeast and NREB in Penicillium, ASD4 does not appear to be involved in regulation of nitrogen metabolism. An Asd-4 null mutant obtained by the rip procedure did not show any effect upon nitrogen control, but instead resulted in severe defects in ascus and ascospore genesis. The Asd-4 rip mutant is dominant to Asd-4+. A cross of the Asd-4 mutant with wild-type resulted in fruiting bodies that appeared to be normal macroscopically but which were complete devoid of asci and ascospores. Introduction of the Asd-4+ gene into the Asd-4 rip mutant corrected the defect in ascus and ascospore development in crosses with wild-type. Mobility shift assays demonstrated that ASD4 acts as a sequence-specific DNA binding protein and recognizes DNA fragments that contain GATA core elements. Gel filtration and cross-linking experiments revealed that the ASD4 protein exists as a tetramer in solution. These results suggest that the ASD4 protein functions positively as a transcriptional regulator of sexual development in Neurospora.

Intracellular processing and toxicity of the truncated androgen receptor: nuclear congophilia-associated cell death.

The pathogenesis of the selective motor neuron death in spinal bulbar muscular atrophy (SBMA) is not fully understood. Similar to observations with other mutant polyglutamine (poly Q) expanded proteins, truncated androgen receptor (AR) with expanded poly Q tract cause intracellular aggregates; however, the precise relationship between aggregates and disease pathogenesis is unresolved. In order to have a better understanding of the cellular processing and toxicity of the mutant AR, we focused on a short N-terminal portion of AR containing normal or expanded poly Q repeats, and have carried out biochemical, immunocytochemical, cytochemical and ultrastructural studies of BHK cells at different intervals after transfection. In cells expressing mutant truncated AR, using an anti-AR N-terminal antibody, we observed no immune staining in the nucleus and identified immune negative aggregates surrounded by immunopositive material in the cytoplasm. Congo red staining identified a component of aggregates with a beta-pleated secondary structure in both cytosol and nucleus, while electron microscopy revealed a fibrillary-granular material as the ultrastructural correlate. In addition, acid phosphatase staining and ubiquitin immunocytochemistry demonstrated that in transfected cells, both lysosomal and nonlysosomal degradation systems are actively involved in handling the mutant truncated AR. The temporal relationship of nuclear congophilia to a subsequent massive cell death suggests that entry of proteolytic cleavage products into the nucleus, perhaps the expanded poly Q stretch itself, may play an important role in cell toxicity.

The NIT2 nitrogen regulatory protein of Neurospora: expression and stability of nit-2 mRNA and protein.

In Neurospora crassa, NIT2 is a global transcription factor that positively regulates the expression of up to 100 genes that are related to nitrogen metabolism. NIT2 is responsible for the lifting of nitrogen catabolite repression when the cellular levels of glutamine or other favored nitrogen sources become limited. Immunoprecipitation of the NIT2 protein showed that it is constitutively expressed, although its expression level is elevated a few-fold when nitrate instead of glutamine is used as the sole nitrogen source. The NIT2 protein is very stable and its stability is not affected by the nitrogen sources. The nit-2 transcripts appear to be very stable as well. The lack of significant regulation of cellular levels of nit-2 mRNA and of NIT2 protein suggests that its interactions with other proteins, e.g., in the nitrate assimilation pathway, with NIT4 and NMR, or post-translational modification of NIT2, may play important roles in modulating the function of NIT2 in response to environmental stimuli.

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa.

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.

Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi.

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi. areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF- mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production.

Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa.

Multiple GATA factors - regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence - exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2. controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBSI of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre+ and sre- strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre- mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.

Analysis of a distal cluster of binding elements and other unusual features of the promoter of the highly regulated nit-3 gene of Neurospora crassa.

In Neurospora crassa, the expression of the nit-3 gene (nitrate reductase) is dependent upon nitrogen derepression and nitrate induction and is regulated by two positive-acting transcription factors, NIT2 and NIT4, and a negative regulator, NMR. The presence of a tightly linked cluster of NIT2 and NIT4 binding sites suggested that their close spacing might be required for a synergistic interaction of the NIT2 and NIT4 proteins. We show here that the NIT2 and NIT4 binding sites can be separated without affecting either the expression level or the precise regulation of the nit-3 gene. Studies conducted on the NIT2 site II, which contains only a single GATA element and yet plays a major role in nit-3 gene expression, showed that nucleotides both 5' and 3' of the GATA sequence were important for strong DNA binding in vitro and its activation function in vivo. The nit-3 promoter contains two long AT-rich sequences, one of which is located just upstream of the transcription start sites and is required for optimal promoter function. The nit-3 transcript contains eight TACC repeats in its 5' noncoding region which appear to be involved in mRNA instability. Deletion of these TACC repeats led to a significant increase in the stability of nit-3 mRNA.

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa.

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.

Interaction between major nitrogen regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is required for their synergistic activation of gene expression in Neurospora crassa.

In Neurospora crassa, the major nitrogen regulatory protein, NIT2, a member of the GATA family of transcription factors, controls positively the expression of numerous genes which specify nitrogen catabolic enzymes. Expression of the highly regulated structural gene nit-3, which encodes nitrate reductase, is dependent upon a synergistic interaction of NIT2 with a pathway-specific control protein, NIT4, a member of the GAL4 family of fungal regulatory factors. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter, but, in addition, we show that a direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of the nit-3 structural gene. Neurospora possesses at least five different GATA factors which control different areas of cellular function, but which have a similar DNA binding specificity. Significantly, only NIT2, of the several Neurospora GATA factors examined, interacts with NIT4. We propose that protein-protein interactions of the individual GATA factors with additional pathway-specific regulatory factors determine each of their specific regulatory functions.

Synthesis and differential turnover of the CYS3 regulatory protein of Neurospora crassa are subject to sulfur control.

The transcription factor CYS3 of Neurospora crassa is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism. Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner. cys-3 expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited. cys-3 transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h. CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth. However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available. Upon sulfur repression, cys-3 transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min. These results suggest that a selective elimination of CYS3 is a highly regulated process. Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability. The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes.

Novel compound heterozygous laminina2-chain gene (LAMA2) mutations in congenital muscular dystrophy. Mutations in brief no. 159. Online.

The laminina2-chain gene (LAMA2) encodes a basal lamina protein, laminina2, known to be deficient in one form of congenital muscular dystrophy (CMD). In a laminina2 deficient-CMD patient, we screened the entire LAMA2 cDNA (953bp) by reverse transcriptase polymerase chain reaction combined with single strand conformational polymorphism analysis. Direct sequencing of aberrant conformers in this patient revealed two loss-of-function mutations, consistent with autosomal recessive inheritance. The patient had two novel heterozygous mutations: 1) an exon 4 nonsense mutation caused by a G-->A substitution at cDNA position 547, changing the TGG codon for tryptophan into a TGA stop codon (W166X) in the N-terminus domain VI;ii) an exon 54 frameshift mutation due to a deletion of nucleotide 'C' at cDNA position 7707 (S2553Y), resulting in a premature stop codon (V2587X) in exon 55 in the globular G domain of laminina2 at the C-terminus. These mutations cause a disruption of the open reading frame of LAMA2. The absence of laminina2 observed in the patient's muscle biopsy could result from diminished levels of the LAMA2 transcript. Alternatively, the mutations might lead to translation of a truncated laminina2. By either mechanism the phenotype of congenital muscular dystrophy is believed to be the result of disruption of linkage between the extracellular matrix and the dystrophin glycoprotein complex.

Genetic regulation of nitrogen metabolism in the fungi.

In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins. However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression. Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain. Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa. Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

Molecular genetics of sulfur assimilation in filamentous fungi and yeast.

The filamentous fungi Aspergillus nidulans and Neurospora crassa and the yeast Saccharomyces cerevisiae each possess a global regulatory circuit that controls the expression of permeases and enzymes that function both in the acquisition of sulfur from the environment and in its assimilation. Control of the structural genes that specify an array of enzymes that catalyze reactions of sulfur metabolism occurs at the transcriptional level and involves both positive-acting and negative-acting regulatory factors. Positive trans-acting regulatory proteins that contain a basic region, leucine zipper-DNA binding domain, are found in Neurospora and yeast. Each of these fungi contain a sulfur regulatory protein of the beta-transducin family that acts in a negative fashion to control gene expression. Sulfur regulation in yeast also involves the general DNA binding protein, centromere binding factor I. Sulfate uptake is a highly regulated step and appears to occur in fungi, plants, and mammals via a family of related transporter proteins. Recent developments have provided new insight into the nature and control of the enzymes ATP sulfurylase and APS kinase, which catalyze the early steps of sulfate assimilation, and of the Aspergillus enzyme, cysteine synthase, which produces cysteine from O-acetylserine.

Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa.

Nitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys2/Cys2 zinc finger DNA-binding domain. The negative-acting NMR protein interacts via specific protein-protein binding with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2-NMR protein-protein interactions. These results provide compelling evidence that the specific NIT2-NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.

Functional in vivo studies of the Neurospora crassa cys-14 gene upstream region: importance of CYS3-binding sites for regulated expression.

Sulphate transport in Neurospora crassa is achieved by two distinct sulphate permeases, I and II, encoded by the cys-13 and cys-14 genes, respectively. The synthesis of both sulphate permeases is subject to sulphur repression and requires the global positive-acting regulatory protein CYS3, CYS3, a bZIP DNA binding protein, regulates cys-14 expression at the transcriptional level and binds in vitro specifically to three DNA-recognition sites, A, B, and C, in the cys-14 upstream region. In vivo functional analysis of the cys-14 promoter was carried out with 5' deletions and by deletions or mutations of CYS3 DNA-binding sites. The most distal CYS3-binding site, C, located 1.4kb upstream of the transcriptional start site, is necessary and sufficient to mediate strong transcriptional activation by CYS3; moreover, site C was able to function equally well when it was located at variable distances upstream of the cys-14 gene. Site B, located 1 kb upstream, alone is able to support a moderate degree of cys-14 expression. Site A is not required and does not appear to play any functional role in cys-14 expression, even though it is in close proximity to the transcriptional start site. The presence of multiple copies of CYS3-binding elements A or B in the cys-14 promoter results in a parallel increase of regulated gene expression. When a transforming cys-14 gene becomes integrated at ectopic locations in the host genome, it can be expressed in an unregulated fashion, presumably by coming under the control of other promoter elements. Our results also suggested that at least one enzyme in the sulphate catabolic pathway requires a functional CYS3 protein for expression.

Determination of the Neurospora crassa CYS 3 sulfur regulatory protein consensus DNA-binding site: amino-acid substitutions in the CYS3 bZIP domain that alter DNA-binding specificity.

CYS3 is the positive-acting global regulatory protein involved in the sulfur control circuit in Neurospora crassa and belongs to the family of bZIP DNA-binding proteins. Here we report a characterization of native DNA-binding sites recognized by CYS3. DNA footprinting experiments and systematic mutational analysis were used to define the consensus CYS3-binding sequence, 5'-ATGPuPyPuPyCAT, a 10-bp palindrome. The sequence 5'-ATGACGTCAT acts as a strong binding site, and all single nucleotide changes within this sequence resulted in a reduction, or even complete loss, of CYS3 DNA-binding. Site-directed mutagenesis was employed to study two uncharged residues, serine 113 and phenylalanine 116, in the basic region of the CYS3 protein bZip DNA-binding domain. Ser113 appears to be directly involved in a specific interaction with nucleotide 2 of the binding site, possibly by making a direct contact with this base, and Phe116 contributes significantly to DNA-binding affinity.

The regulatory protein NIT4 that mediates nitrate induction in Neurospora crassa contains a complex tripartite activation domain with a novel leucine-rich, acidic motif.

Expression of nit-3 and nit-6, the structural genes which encode nitrate reductase and nitrite reductase in Neurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys6/Zn2 zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo in N. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.

Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa.

The nit-2 gene of Neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation. An E. coli-expressed NIT2/beta-Gal fusion protein binds specifically to DNA in vitro by recognizing GATA core elements. Nuclear extracts prepared from a wild-type N. crassa strain contain a protein factor which displays all of the properties expected for the native NIT2 protein. The native NIT2 protein in nuclear extracts binds with high affinity to DNA fragments which contain two GATA elements, weakly to fragments with a single GATA element, and fails to bind to DNAs which lack these sequences. The DNA binding ability of the protein factor in nuclear extracts is efficiently blocked by a polyclonal antibody developed against the zinc-finger region of NIT2 protein. Western blot analysis with the anti-NIT2 antiserum revealed a specific protein with a size of approximately 110,000 daltons, in excellent agreement with the predicted size of NIT2. Both the specific NIT2 DNA binding activity and the protein detected by Western blot are totally lacking in nuclear extracts of a nit-2 rip mutant strain. These results all support the conclusion that the native NIT2 protein in Neurospora cells has been identified. The NIT2 protein is localised in nuclei and could not be detected in the cytoplasmic fraction of cells subjected to nitrogen derepression or nitrogen repression, indicating that the nuclear import of NIT2 is not regulated.

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase.

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.