Current Genetics

Current Genetics (1984, 8:581-588

© Springer-Verlag 1984

Biochemical characterization of the molybdenum cofactor mutants of Neurospora crassa: in vivo and in vitro reconstitution of NADPH-nitrate reductase activity Nigel Stuart Dunn-Coleman 1

International Plant Research Institute, San Carlos, California 94070, USA

Summary. Molybdenum cofactor (MoCo) mutants of Neurospora crassa lack both NADPH-nitrate reductase and xanthine dehydrogenase activity. In vivo and in vitro studies to further characterize these mutants are now reported. The MoCo mutants nit-9A and nit-9B are capable of growing, albeit poorly, with nitrate as the sole nitrogen source, provided high levels of molybdate are present. The MoCo mutants nit-9A, nit-9B and nit-9C, but not nit-l, nit-7 or nit-8, have significant levels of NADPH-nitrate reductase when grown in nitrate medium containing 30 mM molybdate. In vitro reconstitution experiments using cell free extracts of the N. crassa MoCo mutants and E. coli HB101 as a source of wildtype MoCo were performed. MoCo from E. coli was capable of reconstituting NADPH-nitrate reductase activity to nit-l, nit-7 and nit-8. Molybdate is required for the in vitro reconstitution of NADPH-nitrate reductase activity. It was not possible to in vitro reconstitute NADPH-nitrate reductase activity in the MoCo mutants nit-9A, nit-9B or nit-9C. Key words: Nitrate reductase - Reconstitution

Introduction

Nitrate assimilation in Neurospora crassa occurs in two steps: the reduction of nitrate to nitrite by NADPHnitrate reductase and the reduction of nitrite to am-

1 Present address: Visiting Scientist, Central Research and Development Department, Du Pont Experimental Station, Wilmington, Delaware 19898, USA

monium by NAD(P)H-nitrite reductase. The ammonium produced is then used to generate glutamate and glutamine by the sequential action of glutamate dehydrogenase and glutamine synthetase. Nitrate reductase is believed to be a homodimeric protein with a molecular weight of possibly 270,000 (Homer 1983), although other workers have previously determined its molecular weight to be 228,000 (Garrett and Nason 1969). In 1970, Nason and his collaborators (Nason et al. 1970) reported that NADPH: nitrate reductase activity could be reconstituted by combining extracts of a particular nitrate reductase defcient strain, nit-l, with extracts of the nit-3 mutant strain or wild-type extract. Subsequent experiments (Ketchum et al. 1970; Nason et al. 1971) gave evidence that a molybdenum cofactor was provided by the wild-type or other extracts to the nit-1 extract and this endogenous molybdenum cofactor (MoCo) complemented apo-nitrate reductase in the nit-1 to form active NADPH-nitrate reductase. These observations have led to an understanding of the nature of the structure of nitrate reductase. The nit-1 mutant contained a NADPH-cytochrome c reductase (diaphorase) activity displaying a sedimentation coefficient of 4.5 (4.55). This activity increased in nit-1 mycelium grown with nitrate (Nason et al. 1970) and is cross reactive to anti-nitrate reductase antibodies (Amy and Garrett 1979). The conclusion drawn by Nason et al. (1970) was that this NADPH-cytochrome c reductase is the apoprotein nitrate reductase, i.e. molybdenum - free enzyme subunits and that, in the presence of a low molecular weight (< 1,000 daltons) MoCo, these apoprotein subunits dimerize to yield active NADPH-nitrate reductase (8S). In addition, it has been recently confirmed that the reconstituted enzyme exhibits properties similar to that of the wild-type enzyme (Homer 1983). In N. crassa, MoCo is used by two enzymes, nitrate reductase and xanthine dehydrogenase. In consequence,

582

N.S. Dunn-Coleman: Molybdenum cofactor mutants ofN. crassa

mutants defective in MoCo synthesis cannot utilize nitrate of purines such as hypoxanthine as a nitrogen source (Coddington 1976). Recently, Tomsett and Garrett (1980, 1981) have undertaken an extensive biochemical and genetical study of nitrate assimilation in N. crassa and have isolated and characterized several new loci affecting MoCo synthesis in N. crassa. Table 1 summarizes nitrate assimilation mutants in N. crassa, other filamentous fungi and Nicotiana. Tomsett and Garrett found six complementation groups (nit-9ABC, nit-l, nit-7 and nit-8) defining four genetic loci, the nit-9ABC locus being possibly a single gene comprised of three cistrons or two contiguous genes, nit-9A and nit-9C, with nit-9B being a double mutant. Similar mutants have been found in both Aspergillus nidulans and Penicillium chrysogenum (see Table 1). Arst et al. (1970) demonstrated that mutants at the cnxE locus in A. nidulans were molybdaterepairable, since high concentrations of exogenous molybdate partially restored both nitrate reductase and xanthine dehydrogenase activity. Similar molybdaterepairable mutants have been found in P. chrysogenum and in Nicotiana (see Table 1). MacDonald and Cove (1976) have isolated temperature-sensitive mutants of cnxE, cnxF and cnxH loci in A. nidulans, but only cnxH temperature sensitive mutants have a thermolable nitrate reductase activity. They tentatively concluded that the cnxH gene product may have a structural role in the nitrate reductase molecule. However, the function of any MoCo locus is presently unknown; it is thought that they encode enzymes concerned with molybdenum uptake and MoCo biosynthesis. Arst et al. (1981) isolated mutants of a sixth locus cnxJ. Mutants at this locus have reduced MoCo levels and Arst et al. speculated that the cnxJ locus functioned in MoCo regulation. Garrett and Cove (1976) understook a biochemical study of the MoCo mutants of A. nidulans by in vitro complementation. They found that the co-homogenization of mycelium from a niaD16 structural gene mutant with mycelium from either cnxA6, cnxE29, cnxF12, cnxG4 or cnxH3 resulted in the formation of NADPHnitrate reductase activity. Similar results were obtained with the nit.3 nitrate reductase apoprotein mutant and the MoCo mutants nit-l, nit-7, nit-8 and nit-9ABC in N. crassa (Tomsett and Garrett (1981). In both A. nidulans and iV. crassa it has not been possible to reconstitute NADPH-nitrate reductase activity by the cohomogenization of different MoCo mutants (Garrett and Cove 1976; Tomsett and Garrett 1981). Two types of MoCo mutant have now been isolated in Nicotiana tobacurn (see Table 1). Mutants in both the cnxA and cnxB locus lack both nitrate reductase activity and xanthine dehydrogenase activity. However, only in cnxA mutants will molybdate partially restore nitrate reductase and xanthine dehydrogenase activity. In addition, MoCo from cnxA but not cnxB is capable of

restoring NADPH-nitrate reductase activity to the N. crassa mutant nit-1. Nitrate reductase structural gene and MoCo mutants have recently been isolated in Nicotiana plumbaginifolia (Marton et al. 1982). The cnxA mutant (formally NX1) was shown to be molybdate repairable. Xuan et al. (1983) have used interspecific hybridizations involving four cnx mutants of N. plumbaginifolia and N. tobacum and found NX1 and NX9 (now cnxA) to be analagous to cnxA of N. tobacum, NX24 (now cnxB) to be analagous to cnxB ofN. tobacum and NX21 (now cnxC) to represent a third cnx locus. The enzymes of nitrate assimilation are subject to two control mechanisms: Induction by nitrate/nitrite and nitrogen metabolite repression (see Table 1). Nitrate/ nitrite induction is believed to be mediated by a positively acting regulatory gene, encoded by the nit-4~5 locus of N. crassa (or the nirA locus of A. nidulans and P. chrysogenum) which is stimulated by the presence of nitrate or nitrite. Nitrogen metabolite repression prevents the expression of the nitrate assimilation pathway in the presence of reduced nitrogen such as ammonium or glut-amine. Glutamine is believed to prevent the action of a second positive regulatory gene, nit-2 in N. crassa (areA in A. nidulans). It is not known if glutamine acts directly by inactivating the nit-2 gene product (Grove and Marzluf 1981) or indirectly via glutamine synthetase (Dunn-Coteman and Garrett 1980). Garrett and Cove (1976) studied MoCo regulation in A. nidulans. They found that MoCo levels, although not elevatable by nitrate (indicating a lack of involvement of the nirA gene), were lower when A. nidulans was grown with ammonium. Mendel et al. (1981, 1982a, b) studied MoCo regulation in N. tobacum. MoCo content was determined by the ability of a cell-free extract of N. tobacum to restore NADPH-nitrate reductase activity to an extract of N. crassa nit-1. Mendel et al. found that the addition of nitrate to an amino acid-grown culture of wild-type cells resulted in a concurrent increase in both nitrate reductase and MoCo content of the cells. This result was unlike that found by Garrett and Cove (1976) in A. nidulans, when nitrate did not influence MoCo content. However, Mendel et al. (1982a, b) assayed total MoCo-containing extracts. Amy and Rajagopalan (1979)and Amy (1980) have extensively studied MoCo production in both wildtype and MoCo mutants of E. coll. MoCo from E. coli was shown to be capable of reconstituting NADPH-nitrate reductase activity to cell free extracts of the N. crassa mutant nit-1. In vivo and in vitro to further characterize the MoCo mutants of N. crassa are now reported. High levels of molybdate in growth medium partially repair the NADPHnitrate reductase activity in nit-9A,B,C mutants. In vitro reconstitutions using E. coli cell-free extracts as a source of MoCo, are capable o f restoring NADPH-nitrate reductase activity to the MoCo mutants nit.l, nit-7 and nit.8.

Table 1. Nitrate assimilation mutants in fungi and Nicotiana N. erassa

A. nidulans

P. ehrysogenum

N. tobacum

N. plumbaginifolia

Function

Reference

nit-3

niaD

niaA

nia

nia

Nitrate reductase apoprotein structural gene

For review ofN. crassa, see Tomsett and Farrett (1980, 1981)

nit-6

niiA

niiA

Nitrite reductase apoprotein structural gene

For reviews ofA. nidulans, see Cove (1976a, b, 1979); Kinghorn and Pateman (1977); Pateman and Kinghorn (1977), and Tomsett and Cove (1979). P. chrysogenum work by Birkett and Rowlands (1981)

nit4~5

nirA

nirA

trans-acting regulatory gene required for the expression of nitrate reductase and nitrite reductase under conditions of induction by nitrite

For N. tobacum, see Mendell et al. (1981, 1982a, b); Buchanan and Wray (1983). ForN. plumbaginifolia, see Marton et al. (1982); Xuan et al. (1983)

nit-1

-

Encodes an enzyme for MoCo synthesis?

nit-2

areA

trans-acting regulatory gene required for the expression of a wide range of ammoniumgenerating enzymes, including nitrate reductase and nitrite reductase

nit-9ABC

cnxABC

cnxABC

-

cnxD

-

-

Encodes an enzyme for MoCo synthesis?

cnxE

cnxE

cnxA

cnxA

c n x E mutants ofA. nidulans, P. chrysogenum and c n x A mutants ofN. tobacum, N. plumbaginifolia are molybdate repairable

cnx B

cnxB

Encode enzymes for MoCo biosynthesis?

enxC

Encode enzymes for MoCo biosynthesis?

cnxF

Possibly a single gene with 3 intracistronic complementational groups or 2 contiguous genes c n x A and cnxC, with c n x B mutants being a double loss class

cnxF*

Encode an enzyme for MoCo biosynthesis?

cnxH cnxG

m

Structural component of MoCo? enxG *

Encode an enzyme for MoCo biosynthesis?

cnxJ

Possible regulatory locus, affecting MoCo levels

nit-1

Encodes an enzyme for MoCo synthesis?

nit-7

Encodes an enzyme for MoCo synthesis?

nit-8

m

crnA

Molybdenum cofactor (MoCo) loci. Mutations in any one of these loci leads to a loss of nitrate reductase, purine hydroxylase I and purine hydroxylase II and activity

_

-

_

041

Arst et al. (1982a)

Encodes an enzyme for MoCo synthesis? Nitrate permease locus

crnA, Tomsett and Cove (1979); Brownlee and Arst (1983). 041, Queshi et al. (1982)

* It is not known if the nomenclature used with the P. chrysogenum cnx mutants is strictly equivalent to the c n x F and c n x G mutants ofA. nidulans

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N.S. Dunn-Coleman: Molybdenum cofactor mutants ofN. crassa

Materials and methods

nidulans conidia were transferred onto solid minimal media (Cove 1966) using a sterile needle, supplemented with the appropriate nitrogen source and the plates were incubated for several days at 30 °C before scoring for the degree of growth.

Strains. The standard wild-type strains of Neurospora crassa, were Oak Ridge strains 74-OR8-1a and 74-OR23-1A. The mutant strains nit-1 (34547), nit-7 (V1M59), nit-8 (V1M44), nit-9A (V1M5), nit-9B (V1M32), nit-9C (V1M50) were obtained from the Fungal Genetics Stock Center, Humboldt State University Foundation, Arcata, CA. Aspergillus nidulans. The strain used as the wild-type was a translocation-free biotin auxotroph bi-1 (Glasgow No. 051). The following mutant strains were provided by Dr. J. Clutterbuck from the Glasgow University, Scotland, Stock Collection, niaD17 (No. 9126), cnxA5 (No. 9831), c n x B l l (No. 9055), cnxC3 (No. 9053), cnxE14 (No. 9059), cnxF8 (No. 9713), cnxG4 (No. 9614), cnxH4 (No. 9063). Escherichia coil HB101 was used as the wild-type strain. Media and routine procedures. The media and routine manipulation procedures for N. crassa have been described by Davis and DeSerres (1970), Tomsett and Garrett (1980) anf for A. nidulans by Cove (1966), modified after Pontecorvo et al. (1955). E. coli procedures described by Maniatis et al. (1982) were used. Growth conditions. N. crassa mycelia to be used for enzyme assays were grown for 40 h at 25 °C on Vogel's minimal medium (Davis and DeSerres 1970) with 80 mM NH4C1 + 2.5 mM glutamine and then transferred to fresh medium containing 20 mM NaNO 3 as the nitrogen source for 5 h at 25 °C. The mycelia were harvested by filtration on Buchner funnels, washed several times with distilled water and frozen at - 7 0 °C used.

In vitro reconstitution o f N. crassa nitrate reductase activity using E. coli cell-free extract. N. crassa mycelia were homogenized in 100 mM phosphate, 5 mM EDTA, 1% NaC1, 1 mM t~-mercaptoethanol [pH 7.3 ] (Garrett and Cove 1976). The crude homogenate was then centrifuged for 20 min at 27,000 x g at 4 °C to give a supernatant which was used as a crude extract. E. coli cultures were grown overnight at 37 °C in 500 mls TSB medium with 1 mM NaMoO 4. The culture was then centrifuged for 20 min at 27,000 g at 4 °C and the bacterial pellet resuspended in 10 mls o f 10 mM phosphate, 5 mM EDTA (pH 7.5). The bacterial suspension was then sonicated with a Brinkman Sonicator for 3 x 30 second periods, cooling the suspension on ice in between conications. The suspension was then centrifuged for 20 min at 27,000 g at 4 °C. The supernatant was designated as the crude extract. E. coli crude extracts were assayed for the presence of molybdenum cofactor by a complementation assay using a crude extract from one of the N. crassa mutants nit-l, nit-7 or nit-8. Usually 5-100/~I of E. coli extract and 100 ~zlN. crassa extract were added to either Buffer A (10 mM phosphate, 0.5 mM EDTA and 20 mM NaMoO 4 pH [7.41) or Buffer B which lacked NaMoO 4 (10 mM phosphate, 0.5 mM EDTA pH [7.4]) to a final volume of 250 /A. This incubation mixture was maintained at room temperature; 75 ~zl samples were taken from the incubation mixture at time 0, 10 min and 20 rain to determine nitrate reductase activity. Protein determinations. The method of Lowry et al. (1951) was used, with bovine serum albumin as the standard.

Enzyme assays. Nitrate reductase was assayed according to the diazo-coupling procedure described by Nason and Evans (1953) as modified by Garrett and Nason (1967). A solution of freshly prepared Na2SO 3 was added to the assay mixture at a concentration of 5 mM to inhibit nitrite reductase activity (Vega et al. 1975).

Results

Growth tests. N. crassa, conidial suspensions were made from each of the strains to be tested and then "spotted" onto solid Fries Minimal Medium (Davis and DeSerres 1970) supplemented with the appropriate nitrogen source. The plates were incubated for several days at 25 °C before scoring for degree of growth. A.

o n the u t i l i z a t i o n o f n i t r a t e b y N. crassa M o C o m u t a n t s ,

G r o w t h tests. Aspergillus nidulans Prior t o c h a r a c t e r i z i n g t h e possible e f f e c t o f m o l y b d a t e t h e M o C o m u t a n t s o f A . nidulans were p l a t e d o n t o m e d i a c o n t a i n i n g nitrate as the sole n i t r o g e n source,

Table 2. Growth responses of wild-type and mutant strains ofA. nidulans NH4C1 + KNO 3

Wild-type enxA cnxB cnxC enxE cnxF cnxG cnxH

4 4 4 4 4 4 4 4

KNO 3 + Molybdate 1 ~M

1 mM

5 mM

33 mM

50 mM

4 0.5 0.5 0.5 0.5 0.5 0.5 0.5

4 0.5 0.5 0.5 0.5 0.5 0.5 0.5

3 0.5 0.5 0.5 3 0.5 0.5 0.5

2 0.5 0.5 0.5 2 0.5 0.5 0.5

2 0.5 0.5 0.5 2 0.5 0.5 0.5

The growth tests were carried out at 25 °C on appropriate supplemented minimal medium. 0.5-4 indicates increasing levels of growth. The carbon source was D-glucose (1% w/v). The nitrogen source concentration was 10 mM NH4C1 + 10 mM KNO3/or 20 mM KNO 3

N. S. Dunn-Coleman: Molybdenum cofactor mutants ofN. crassa

585

Table 3. Growth responses of wild-type and mutant strains of N. erassa 10 mM NHaC1

Wild-type nit-1 nit-7 nit-8 nit-9A nit-9B nit-9C

10 mM KNO 3 + Molybdate

4 4 4 4 4 4 4

35 gM

1 mM

5 mM

10 mM

25 mM

35 mM

50 mM

4 0.5 0.5 0.5 0.5 0.5 0.5

4 0.5 0.5 0.5 0.5 0.5 0.5

4 0.5 0.5 0.5 0.5 0.5 0.5

4 0.5 0.5 0.5 0.5 0.5 0.5

4 0.5 0.5 0.5 3 3 0.5

3 0 0.5 0.5 3 2 0.5

3 0 0.5 0.5 2 1.5 0.5

The growth tests were carried out at 25 ° C on appropriate supplemented minimal medium. 0.5-4 indicate increasing levels of growth. The carbon source was sucrose (2% w/v)

Table 4. The effect of molybdate on in vivo nitrate reductase activity in wild-type and mutant strains of N. crassa Nitrate reductase activity

Wild-type nit-1 nit-7 nit-8 nit-9A nit-9B nit-9C

1 #M Mo

30 mM Mo

163

37

3

UD UD 1.4 4.0 2.0

4

UD UD 25 25 32

Mycelia were grown for 40 h at 25 °C in minimal medium (Tomsett and Garrett 1981) with 3% cane sugar plus 80 mM NH4C1 + 2.5 mM glutamine and then transferred to 40 mM KNO 3 with either 1 #M or 30 mM NaMoO 4 for a 5-h period. Enzyme activities are expressed as nmole/10 rain assay/rag protein. UD = undetected activity

with varying amounts o f molybdate. The results of this experiment are shown in Table 2. Only the cnxE mutant showed repair o f growth when grown with high levels o f molybdate. These results confirm those o f Arst et al. (1970). In fact, although Arst et al. reported that 33 mM molybdate would repair growth of c n x E mutants, as little as 5 mM molybdate was sufficient to enhance the growth o f the enxE mutant on nitrate.

Neurospora crassa

The N. crassa MoCo mutants were growth tested on media containing 10 mM KNO3 as the sole nitrogen source with varying concentrations o f molybdate. Surprisingly, only the nit-9A and nit-9B mutants showed enhanced growth with high amounts o f molybdate ad-

ded to the medium (see Table 3). To remove the possibility that the strains had been mislabeled, additional strains were sent for from the N. erassa Fungal Genetics Stock Center. Identical results were obtained from these cultures; only nit-9A and nit-9B showed enhanced growth at high molybdate concentrations. The c n x A B C locus o f A. niclulans which is believed to be analagous to that o f nit-9ABC of N. crassa showed no molybdate repair of growth, when grown with nitrate (see Table 2). I f therefore appears no mutants o f N. crassa have y e t been isolated that are equivalent to the enxE mutants o f A. n idulans.

Molybdate repair o f nitrate reductase activity in N. erassa

The results with the growth tests of the MoCo mutants nit-9A and nit-9B indicated that molybdate could restore the mutant's growth when nitrate was the nitrogen source. To confirm that molybdate was in fact restoring nitrate reductase activity, wild-type and the MoCo mutants o f N. erassa were grown in the presence or absence o f 30 mM molybdate with nitrate as the sole nitrogen source and the levels o f nitrate reductase activity determined. The results are shown in Table 4. Growth with nitrate and 30 mM molybdate reduced the lebel of nitrate reductase in wild-type as compared to that obtained after growth with only 1 mM molybdate. This result may be a related to the somewhat inhibitory effect 30 mM molybdate has on growth of wild-type iV. crassa. The mutants nit-l, nit-7 and nit-8 showed no molybdate repair of their nitrate reductase activity. These results parallel those obtained from growth tests on the mutant strains (Table 3). All three nit-9 mutants showed molybdate repair of nitrate reductase, resulting in levels of nitrate reductase approaching when those obtained with wild-type grown on 30 mM molyb-

586

N.S. Dunn-Coleman: Molybdenum cofactor mutants ofN. crassa

Table 5. In vitro reconstitution of nitrate reductase activity in N. crassa molybdenum cofactor mutants by molybdenum cofactor from wild-type E. coli

reductase activity was found when nit-9A, nit-9B or nit-9C was incubated with E. coli cell-free extracts (results not shown). In the previous experiments (see Table 4), growth of nit.l, nit.7 or nit-8 in the presence of high levels of molybdate did not lead to repair of NADPH-nitrate reductase activity. However, NADPH-nitrate reductase activity in these mutants can be restored when wild-type E. coli cell-free extracts provide a source of MoCo. This suggests that nit-l, nit-7, and nit-8 synthesize a defective MoCo which does not associate with, or freely dissociates from the nitrate reductase apoprotein, and that MoCo from E. coli can replace the mutant's defective MoCo to restore NADPH-nitrate reductase activity. The nit-9A, nit-9B and nit-9C mutants appear to synthesize a defective MoCo which lacks the molybdenum ion. Growth of these mutants with high levels of exogenous molybdate will overcome the mutant's defect. However, the absence of in vitro reconstitution of NADPH-nitrate reductase activity in these MoCo mutants suggests that the nit-9A, nit-9B and nit.9C mutants synthesize a MoCo which associates with the nitrate reductase apoprotein and cannot be displaced by MoCo from E. coli.

Nitrate reductase activity

Wild type nit-1 nit-7 nit-8 nit-gA nit-9B nit-9C

NoMo

20 mM Mo

95 1.1 0.8 UD UD UD UD

56 52 44 27 UD UD UD

N. crassa mycelia were grown for 40 h at 25° in minimal medium with 3% cane sugar plus 80 mM NH4C1 and transferred to 40 mM KNO3 for a 5 h period. E. coli HB101 was grown overnight at 37 °C in 3% TSB medium. Cell-free extracts were isolated from N. crassa and E. coli (see Materials and methods) and 100 t~IN. crassa cell-free extract was incubated with 50 Izl E. coli cell-free extract and 100 pl 10 mM PO4 Buffer [pH 7.4], 0.5 mM EDTA with or without 20 mM NaMoO4, for 10 rain at room temperature. NADPH-nitrate reductase activity was then determined. Results are expressed as nmole NO~- formed/10 rain assay/rag protein. UD = undetectable activity

date. Molybdate repair of nitrate reductase in nit-9A and nit.9B agrees with the enhanced growth observed for these mutants when grown on molybdate supplemented nitrate medium (see Table 3). Molybdate repair o f nitrate reductase in nit-9C was surprising since growth o f this on nitrate was not enhanced by molybdate. These conflicting observations suggest that the target for molybdate's inhibitory effect on growth is not nitrate reductase, and underscores the difficulty in interpreting growth tests at high molybdate concentrations.

In vitro repair o f nitrate reductase activity, using E. coli cell-free extracts as a source o f MoCo

To characterize the N. crassa MoCo mutants further, experiments were performed using wild-type E. coli (HB101) cell-free extracts as a source of MoCo in attempts to reconstitute NADPH-nitrate reductase activity in vitro. The results are shown in Table 5. NADPH-nitrate reductase activity was restored in nit-1 after incubation with wild-type E. coli cell-free extracts; however, in vitro reconstitution of activity requires the presence of molybdate in the incubation buffer. These results are similar to those of A m y and Rajagopalan (1979). In addition, nit-7 and nit-8 were also found to be capable o f in vitro reconstitution of NADPH-nitrate reductase when incubated with E. coli cell-free extracts. However, no in vitro reconstitution of NADPH-nitrate

Discussion Nitrate reductase, xanthine dehydrogenase, sulfite oxidase, formate dehydrogenase and aldehyde oxidase from a wide range of phylogenetic sources contain a molybdenum cofactor (MoCo). MoCo, when released from these enzymes, can displace in vitro the defective MoCo o f the N. crassa nit-1 mutant to reconstitute N. crassa NADPH-nitrate reductase activity. Recently, Johnson et al. (1980) have shown this MoCo to be a novel pterin (molybdopterin). Although the enzymes involved in the synthesis of MoCo are unknown, mutants from a variety o f organisms show similar characteristics. MoCo mutants in filamentous fungi andNicotiana (the most biochemically characterized plant MoCo mutants) both lack nitrate reductase and xanthine dehydrogenase activity. In addition, the enxE mutants of A. nidulans and P. chrysogenum and the c n x A mutants o f N . tobacum and N. plumbaginifolia have a molybdate-repairable nitrate reductase. When these mutants are grown with high levels exogenous molybdate the levels of both nitrate reductase and xanthine dehydrogenase activity are partially restored. The chlD mutant of E. coli shows similar restoration of nitrate reductase activity when grown with high levels of molybdate (Sped and DeMoss 1975). The behavior of these mutants, can be explained if they are defective in the insertion of molybdate into the MoCo. Thus high levels of molybdate in the medium partially overcome this defect.

N. S. Dunn-Coleman: Molybdenum cofactor mutants of N. crassa

587

Only the cnxE mutant of A. nidulans was found to have molybdate repairable growth on nitrate (Arst et al. 1981). Subsequently, the cnxA mutants ofN. tobacum (Mendel et al. 1981), N. plumbaginifolia (Marton et al. 1982) and cnxE of P. chrysogenum showed similar molybdate-enhanced growth on nitrate. The cnx m u t a n t s o f Arabidopsis thaliana (Braaksma and Feenstra 1982) and I2D12 mutants of Hyoscyamus muticus (Gebhardt et al. 1982) also respond similarly to molybdate. The nit-9A and nit-9B mutants were found to show enhanced growth on nitrate when high levels of molybdate were present in the medium (Table 2). In addition, nit-9A, nit-9B and nit-9C were found to have substantially restored NADPH-nitrate reductase activity when mycelia were grown on nitrate with 30 mM molybdate (Table 3). The nit-9ABC locus genetically appears directly analogous to the cnxABC locus of A. nidulans. It was, therefore, surprising that mutants of the nit-9ABC locus showed molybdate-repairable nitrate reductase, when cnxABC mutants fail to show any molybdate repair of growth on nitrate. In vitro reconstitution studies using the N. crassa MoCo mutants and E. coli cell-free extracts as a source o f wild-type MoCo have provided insights into the MoCo defect in the N. crassa mutants. Apparently, E. coli MoCo can replace the defective MoCo in nit-1 (confirming Amy and Rajagopalan's results, 1979), nit-7 and nit.8 to reconstitute NADPH-nitrate reductase activity to these mutants. Thus the defective MoCo apparently does not associate with the nitrate reductase apoprotein, or can be freely dissociated. The nit-l, nit-7 and nit-8 mutants of N. crassa showed no molybdate repair of growth on nitrate or reconstitution of NADPH-nitrate reductase activity. These mutants are, therefore, unlike cnxE of A. nidulans which has molybdate-repairable growth on nitrate and restored NADPH.nitrate reductase activity. The nit-9ABC mutants, when grown with high levels of exogenous molybdate, had substantial levels of NADPH-nitrate reductase activity. In this respect, the nit-9ABC mutants are similar to cnxE ofA. nidulans. The mutant nit-1 has most frequently been used as the source of apoprotein for in vitro complementation experiments using MoCo from various sources. The results in this communication show that nit-7 and nit-8 can also be used for the in vitro reconstitution o f NADPHnitrate reductase.

plant transformation experiments. Several N. crassa genes have been cloned on the basis of their ability to transform E. coli mutant to the wild-type phenotype. For example, the qa-2 gene of N. crassa (encoding 5dehydroquinate hydrolyase) was cloned by Vapnek et al. (1977) due to its ability to transform an aroD mutant o f E. coli. The biochemical characterization ofN. crassa MoCo mutants described here was undertaken in an effort to identify an N. crassa mutant equivalent to the cnxE gene of A. nidulans, cnxA ofN. tobacum, or chlD o f E. coli. The following paper (Dunn-Coleman 1984) describes the cloning and preliminary characterization o f a MoCo gene from N. crassa.

Because of the apparent conservation in MoCo structure and the similarity of MoCo mutants, in particular the cnxA mutants of N. tobacurn and cnxE mutants of A. nidulans, it is possible that these mutants are defective at the same step in MoCo biosynthesis. Cloning the N. crassa MoCo gene corresponding to cnxE gene of A. nidulans or cnxA gene ofN. tobacum would allow direct molecular approaches for investigating MoCo biosynthesis, in addition to providing useful material for the

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Note added in proof. When the MoCo mutants nit-9A, 9B and 9C are grown at 32 °C with 10 mM KNO 3 + 25 mM molybdate all three MoCo mutants display molybdate repairable growth.

Communicated by K. P. VanWinkle-Swift Received March 19 / June 4, 1984