Nitrate reductase mutants

The idea that the same cofactor species operated in all Mo enzymes originated from a reconstitution assay. In this assay method, the isolated Moco from one enzyme, such as XO, is inserted into a cofactor-free mutant (Nit-1) of nitrate reductase from Neuraspora crassa, where it can reactivate or reconstitute normal nitrate reductase catalytic activity. It is now recognized that the Mo at the active site has many different coordination environments, as has been illustrated for the three Mo families in Fig. 1. In this context, the mutant nitrate reductase assay experiment is interpreted as involving some reprocessing of the inserted molybdenum cofactor from foreign enzymes to obtain the correct form of the cofactor for nitrate reductase catalysis. The Moco designation, if it is to be used, must refer to the family of sites present in Moco enzymes. 

The molybdenum cofactor was liberated from D. gigas AOR, and under appropriate conditions was transferred quantitatively to nitrate reductase in extracts of Neurospora crassa nit-1 mutant) to yield active nitrate reductase 217). On the basis of molybdenum content, the activity observed for reconstitution with molybdenum cofactor of D. gigas was lower (25%) than the values observed for the procedure using extractable molybdenum cofactor of XO, used as reference. This result can now be put in the context of the difference in pterin present (MPT-XO and MCD-AOR) 218). 

The recognition that the Mo in the molybdoproteins exists in organic cofactor forms came from studies of mutants of Aspergillus and Neurospora.650 In 1964, Pateman and associates discovered mutants that lacked both nitrate reductase and xanthine dehydrogenase. Later, it was shown that acid-treated molybdoenzymes released a material that would restore activity to the inactived nitrate reductase from the mutant organisms. This new coenzyme, a phosphate ester of molybdopterin (Fig. 15-17), was characterized by Rajagopalan and coworkers.650 651 A more complex form of the coenzyme, molybdopterin cytosine dinucleotide... 

The assimilatory nitrate reductase from Chlorella contains the molybdenum cofactor, as evidenced by the ability of the enzyme to donate the cofactor to the nitrate reductase of the mutant nit-1 of N. crassa. Reduction of the enzyme with NADH gives the Mov ESR signal, which is abolished on reoxidation with nitrate. Line shape and g values of the signal show a pH dependence similar to those observed previously for sulfite oxidase. The signal observed at pH 7.0 shows evidence for interaction with a single exchangeable proton.1053... 

An organism such as E. coli has at least five molybdoenzymes. It will be of great interest to look at the synthesis, availability and cellular distribution of the molybdenum cofactor and its relationship to the function of these molybdoenzymes at different stages of the cell cycle. The study of chlorate-resistant1057 and nitrate reductase-deficient1058 mutants of E. coli, which are... 

Ch6rel, I., Gonneau, M., Meyer, C., Pelsy, F. Caboche, M. (1990). Biochemical and immunological characterization of nitrate reductase-deficient nia mutants of Nicotiana plumbaginifolia. Plant Physiology 92, 659-65. 

Dorbe, M.-F., Caboche, M. Daniel-Vedele, F. (1992). The tomato NIA gene complements a Nicotiana plumbaginifolia nitrate reductase-deficient mutant and is properly regulated. Plant Molecular Biology 18,.363-75. 

Nason, A., Lee, K.Y., Pan, S.-S., Ketchum, P.A., Lamberti, A. Davies, J. (1971). In vitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes. Proceedings of the National Academy of Sciences (USA) 68, 3242-6. 

Pelsy, F. Gonneau, M. (1991). Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia. Genetics 127, 199-204. 

Vaucheret, H., Chabaud, M., Kronenberger, J. Caboche, M. (1990). Functional complementation of tobacco and Nicotiana plumbagini-folia nitrate reductase deficient mutants by transformation with the wild-type alleles of the tobacco structural genes. Molecular and General Genetics 220, 468-74. 

It is the (MPT)Mo(0)2 cofactor variant that likely reconstitutes the nitrate reductase activity in the Nit-1 mutant [29,31], Although the (MPT)Mo(0)2 unit could be provided directly by sulfite oxidase, nitrate reductase, and even xanthine... 

The assimilatory enzyme from the mold Neurospora crassa has been intensively studied for over two decades, particularly by Nason and his collaborators. Thus, Nason and Evans (39) identified FAD as a prosthetic group in the enzyme Nicholas, Nason, and McElroy (40) showed that molybdenum was required for the synthesis of nitrate reductase Nicholas and Nason (41) suggested its presence in the enzyme Garrett and Nason (42) showed that a b-type cytochrome (cytochrome 6557) co-purifies with this nitrate reductase and Nason et al. (11) suggested, from in vitro complementation experiments with nitrate reductaseless mutants, that the enzyme consists of at least two components required for activity. These workers have suggested that the electron transfer pathway is ... 

The molybdenum cofactor from Rhodospirillum rubrum is dialyzable and is insensitive to trypsin (9). The cofactor can easily be inactivated by heat. One of the problems in purifying this cofactor is the instability and low yields from purified enzymes and crude extracts. Lee et al. (10) showed that the molybdenum cofactor is stabilized by 0.01M sodium molybdate and that the absence of air adds to the stability. These workers used Mo" labeling to show that the molybdenum from the cofactor is found in activated nitrate reductase from the mutant strain of N. crassa. 

It was important to identify mutant strains with defects in nitrogenase similar to the defect observed in nitrate reductase in the Nit-1 mutant strain of N. crassa (6). It was postulated that such strains would be able to synthesize active component II and an inactive component I that could be activated in vitro by the molybdenum cofactor. Cell-free... 

Several decades ago, the earliest genetic work in molybdenum enzymes identified mutants of two fungi, Aspergillus nidulans (125) and Neurospora crassa (126) that lacked all molybdenum enzyme activities, specifically, nitrate reductase, XDH, and aldehyde oxidase. The mutant N. crassa produces an... 

Figure 13. The reconstitution of apo nitrate reductase from mutant nit-1 by dissociated molybdenum cofactor isolated from different enzymes.

Buc, J., Santini, C.-L., Blasco, F., Giordani, R., C rdenas, M. L., Chippaux, M., Comish-Bowden, A., and Giordano, G., 1995, Kinetic studies of a soluble oP complex of nitrate reductase A from Escherichia coli. Use of various (xP mutants with altered P subunits, Eur. J. Biochem. 234 766n772. 

Lu, G., Lindqvist, Y., Schneider, G., Dwivedi, U., and Campbell, W. H., 1995, Structural studies on com nitrate reductase refined structure of the cytochrome b reductase fragment at 2.5 A, its ADP complex and an active-site mutant and modeling of the cytochrome b domain, J. Mol. Biol. 248 9319948. 

Ratnam, K., Shiraishi, N., Campbell, W. H., and Hille, R., 1995, Spectroscopic and kinetic characterization of the recombinant wild-type and C242S mutant of the cytochrome b reductase fragment of nitrate reductase, J. Biol. Chem. 270 24067924072. 

In the early 1970s Nason and co-workers showed that extracts of nit-1 mutants of Neurospora crassa exhibited nitrate reductase activity when mixed with solutions of other molybdenum enzymes that had been subjected to denaturing conditions (20-23). The isolation and... 

The laser photolysis results on the ET behavior of these mutants have been confirmed by steady-state kinetic measurements [58, 59]. Interestingly, the latter experiments have shown that nonconservative mutations at F65 and E94 not only severely inhibit reactivity with FNR, but also with two other ferredoxin-dependent enzymes, nitrite reductase and nitrate reductase [58]. Apparently, similar structural constraints in their interactions with Fd are also operative in these other enzymes. 

These results clearly indicate that by disrupting the nitrate reductase system, an Ecoli strain suitable for practical hydrogen production can be developed which will not be affected by the presence of nitrate in the utilizable substrate. The availability of a mutant strain enabled us to demonstrate the application of a mutant strain deficient in the narG locus. However, the existence of a constitutively synthesized nitrate reductase was reported, a nitrate reductase Z (10). Indeed, by the cultivation of the strain RK5265 in the presence of 100 mM nitrate, no FHL activity was observed (results not shown). Therefore, the ideal E.coli strain will be constructed by introducing mutation in the structural gene for FDH-N. 

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