Grether-Beck S, Igloi G L, Pust S, Schilz E, Decker K, Brandsch R
Biochemisches Institut, Universität Freiburg, Germany.
Mol Microbiol. 1994 Sep;13(5):929-36. doi: 10.1111/j.1365-2958.1994.tb00484.x.
The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M(r) 30,011, 14,924 and 87,677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hino genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.
从嗜烟节杆菌的分解代谢质粒pAO1中鉴定、克隆并测序了尼古丁脱氢酶（NDH）基因。紧邻该基因簇的是6-羟基-L-尼古丁氧化酶（6-HLNO）基因的起始部分。NDH由分子量分别为30,011、14,924和87,677的三个亚基（A、B和C）组成。它属于具有相似亚基结构的细菌羟化酶家族；它们以钼蝶呤二核苷酸、FAD和铁硫簇作为辅因子。这里提供了细菌羟化酶的首个完整一级结构。每个NDH亚基的序列比对显示与真核黄嘌呤脱氢酶（XDH）的序列相似，但与其他已知的含钼细菌酶不同。基于与XDH的比对推断，最小的亚基（NDHB）携带一个铁硫簇，中等大小的亚基（NDHA）结合FAD，而最大的NDH亚基（NDHC）对应于XDH的钼蝶呤结合结构域。ndh和6-hino基因的表达都需要培养基中存在尼古丁和钼。钨抑制酶活性，但不抑制酶蛋白的合成。该酶在嗜烟节杆菌细胞中以可溶形式和膜相关形式存在。在有钨存在的情况下，膜相关NDH的比例增加。
