Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-05, Vol.88 (10), p.4260-4264
• Main Authors: Maier-Greiner, U.H. (Ludwig-Maximilians-Universitat, Munich, Federal Republic of Germany), Obermaier-Skrobranek, B.M.M, Estermaier, L.M, Kammerloher, W, Freund, C, Wulfing, C, Burkert, U.I, Matern, D.H, Breuer, M
• Format: Article
• Language: English
• Subjects: ABONOS NITROGENADOS; Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Base Sequence; Biochemistry; Biological and medical sciences; CHAMPIGNON; Chromosome Mapping; CIANAMIDAS; CLONACION; CLONAGE; Cloning, Molecular; COMPOSE ORGANOAZOTE; COMPUESTO ORGANICO DEL NITROGENO; CYANAMIDE; Cyanamide - metabolism; DNA; DNA, Fungal - genetics; ENGRAIS AZOTE; Enzymes; Enzymes and enzyme inhibitors; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; Fertilizers; FLORA DEL SUELO; FLORE DU SOL; Fundamental and applied biological sciences. Psychology; Gels; GENE; Gene Expression; GENES; GENETICA; GENETIQUE; HERBICIDAS; HERBICIDE; HONGOS; Hydro-Lyases - genetics; Hydro-Lyases - isolation & purification; Hydro-Lyases - metabolism; Kinetics; Lyases; Mitosporic Fungi - enzymology; Mitosporic Fungi - genetics; Molecular Sequence Data; Molecular Structure; Molecular Weight; MYROTHECIUM; Myrothecium verrucaria; Nitriles; Nitrogen; NUCLEOTIDE; Nucleotides; NUCLEOTIDOS; Oligonucleotide Probes; RESISTANCE AUX PRODUITS CHIMIQUES; RESISTENCIA QUIMICA; Substrate Specificity; UREA; UREE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.10.4260

A protein was purified from crude extracts of the soil fungus Myrothecium verrucaria by gel filtration and hydrophobic chromatography to homogeneity; this protein catalyzed the stoichiometric hydration of the fertilizer cyanamide to urea with high substrate specificity. This cyanamide hydratase (urea hydro-lyase; EC 4.2.1.69) contained zinc and consisted of six identical subunits with Mr = 27,700. It was partially sequenced. The protein as detectable only when the fungus was grown on cyanamide as the sole nitrogen source. Genomic DNA from the fungus was cloned, and the gene encoding the enzyme was mapped with an oligonucleotide probe derived from the amino acid sequence within a 25,800-base-pair DNA region. The subunit of the enzyme is encoded by a 797-base-pair DNA sequence containing a 63-base-pair intron. A cDNA clone containing the intronless gene with an open reading frame encoding a sequence of 244 amino acids expressed the enzyme in active form in Escherichia coli with excellent yield