PYRIDINE NUCLEOTIDE-NITRATE REDUCTASE FROM HANSENULA ANOMALA , A NITRATE REDUCING YEAST

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Abstract
A soluble enzyme system which mediates the reduction of nitrate to nitrite was extracted from sonic-disrupted H. anomala and partially purified by salt fractionation. Either DPNH or TPNH can serve as H donor. One mole of DPNH is oxidized for every mole of nitrite formed. Flavin is a required component of the system; the activity of boiled pig liver juice can be completely replaced by flavine adenine denucleotide (FAD) or partially replaced by flavine mononucleotide (FMN). Riboflavin is inactive. A heavy metal, apparently molybdenum, is required since cyanide and azide but not CO are potent inhibitors. The enzyme requires free sulfhydryl groups for activity, since p-chloromercuribenzoate is inhibitory, but the inhibition can be reversed by either glutathione or cysteine. Ninety-five to 100% of the nitrate-reducing activity of the crude homogenate is found in the particle-free supernatant. The enzyme is inducible, activity being detected in extracts from KNO3 or NH4NO3-grown cells but not in those from an NH4Cl or glycine medium. A survey of the distribution of this enzyme has been made among closely related species of Hansenula, Pichia and Debaryomyces. In all strains studied, the ability to grow on nitrate as a sole N source was correlated with the presence of the enzyme. This is therefore a functional system in vivo.