Maier-Greiner U H, Obermaier-Skrobranek B M, Estermaier L M, Kammerloher W, Freund C, Wülfing C, Burkert U I, Matern D H, Breuer M, Eulitz M
Institut für Biochemie, Ludwig-Maximilians-Universität, Munich, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1991 May 15;88(10):4260-4. 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 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.
通过凝胶过滤和疏水色谱法从土壤真菌疣孢漆斑菌的粗提物中纯化出一种蛋白质，使其达到同质状态；这种蛋白质以高底物特异性催化肥料氰胺化学计量地水合生成尿素。这种氰胺水解酶（尿素水解酶；EC 4.2.1.69）含有锌，由六个相同的亚基组成，Mr = 27,700。对其进行了部分测序。只有当真菌在氰胺作为唯一氮源的条件下生长时才能检测到这种蛋白质。克隆了该真菌的基因组DNA，并用源自25,800碱基对DNA区域内氨基酸序列的寡核苷酸探针定位了编码该酶的基因。该酶的亚基由一个795碱基对的DNA序列编码，其中包含一个63碱基对的内含子。一个含有无内含子基因且开放阅读框编码244个氨基酸序列的cDNA克隆在大肠杆菌中以高产率表达出具有活性形式的该酶。
