Genetic and biochemical studies of nitrate reduction in Aspergillus nidulans

Abstract

In Aspergillus nidulans nitrate and nitrite-induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the 3 enzymes. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitropheny])-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrate reductase and hydroxylamine reductase (which are possibly a single enzyme in A- nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH [reduced nicotinamide denine dinucleotide phosphate]. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. Mutants at 6 gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. Mutants are known at 2 gene loci, nii A and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. Mutants at the nii A locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the nii B gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. The growth and enzyme characteristics of a total of 123 mutants involving 9 different genes indicate that nitrate is reduced to ammonium. Only 2 possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only 2 enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.