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.
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 was 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 795-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.
Maier-Greiner
U H
UH
Institut für Biochemie, Ludwig-Maximilians-Universität, Munich, Federal Republic of Germany.
Obermaier-Skrobranek
B M
BM
Estermaier
L M
LM
Kammerloher
W
W
Freund
C
C
Wülfing
C
C
Burkert
U I
UI
Matern
D H
DH
Breuer
M
M
Eulitz
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003484:Cyanamide / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D013379:Substrate Specificity
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D003484:Cyanamide; D006836:Hydro-Lyases; C048831:nitrile hydratase