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.

Abstract

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.Cyanamide (H2N-G-N) in aqueous solution or in the form of its calcium salt is used as a fertilizer in agriculture. It provides ammonia to the soil by its metabolic conversion. It has the additional advantage of acting as an effective herbicide. Therefore it has to be applied prior to sowing. In view of the present interest in the ecological effects of widely used chemicals, we became interested in the metabolic conversion of this not naturally occurring compound. Chemically, cyanamide belongs to the class of nitriles. In spite of the relatively rare occurrence in nature of compounds containing the nitrile group, enzymes that hydrate this group to the correspondihg amide (nitrile hydratases) have been frequently found in various bacteria (1-5) and also in plants (6). In 1973 Stransky and Amberger (7) EXPERIMENTAL PROCEDURES Growth of Fungi. M. verrucaria (DSM no. 2087) was obtained from the Deutsche Sammlung von Mikroorganismen (Braunschweig, F.R.G.) and grown as described (7) but scaled up. From 4 liters of medium, 10-25 g (fresh weight) of mycelium was harvested after 10 days. The rapidly filtered mycelium was stored at -70°C.Cyanamide Hydratase Assay. The assay is based on the decrease in cyanamide concentration during incubation with the enzyme (7). The enzyme (0.05-0.2 units) was incubated with 20mM cyanamide in 5 mM sodium phosphate buffer (pH 7.7) (total volume = 0.25 ml) at 37°C for 15-60 min, depending on the activity of the enzyme. The cyanamide concentration was determined by a colorometric assay at 530 nm in 20 Al of the incubation mixture before and after enzyme addition (8). One unit of cyanamide hydratase metabolizes 1 ,umol of substrate per min under these conditions. Purification of Cyanamide Hydratase. Twenty grams of the frozen mycelium, after addition of 10%6 (vol/vol)

Two Duplicated Genes DDI2 and DDI3 in Budding Yeast Encode a Cyanamide Hydratase and Are Induced by Cyanamide

“…Based on their protein sequence alignment and analysis, we developed a novel enzymatic assay and confirmed that the DDI2/3 genes encode a protein with CAH activity. Cyanamide hydratase enzymes have not been characterized to the level of many other enzymes, and to date there is only one report on CAH enzymatic activity based on proteins extracted from the fungus M. verrucaria (12). This study reports for the first time real time monitoring of a recombinant cyanamide hydratase reaction, and our recorded K m value for Ddi2/3 (17 mM) is comparable with that of the reported native M. verrucaria CAH (27 mM).…”

“…2C), all belonging to Ascomycota. Among them, only CAH has been demonstrated to have a cyanamide hydratase activity (12,31), and Ddi2/3 and CAH appear to be distantly related (Fig. 2, B and C).…”

“…Recombinant Ddi2 Exhibits Cyanamide Hydratase ActivityTo date, there is only one reported characterization of cyanamide hydratase activity from M. verrucaria cell extract, with a measured K m of 27 mM and high substrate specificity (12). To test our hypothesis that DDI2/3 encodes a cyanamide hydratase and to further characterize this class of enzymes, we cloned the DDI2/3 ORF into plasmid pGEX6P-1 as a GST fusion.…”

“…A protein sequence alignment and analysis reveal that Ddi2/3 is a member of the HD domain metalloprotein superfamily (11) and is homologous to a reported cyanamide hydratase (EC 4.2.1.69) from the soil fungus Myrothecium verrucaria (12,13).…”

Novel thermophilic bacteria producing nitrile-degrading enzymes

“…Brevibacterium (Arnaud et al, 1977), Arthrobacter (Asano et al, 1982), Nocardia (Harper, 1985), Klebsiella (Stalker et al, 1988), Corynebacterium (Li et al, 1992) and Rhodococcus (Langdahl et al, 1996)] and fungi [e.g. Fusarium solani (Harper, 1977), Myrothecium verrucaria (Maier-Greiner et al, 1991) and Fusarium lateritium (Cluness et al, 1993)l.…”