Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans.
Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans.
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DOI：
10.1006/jmbi.1998.2227
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发表时间：
1998-12
影响因子：
5.6
通讯作者：
S. Schenk;A. Hoelz;Beate Krauβ;K. Decker
中科院分区：
文献类型：
--
作者：
S. Schenk;A. Hoelz;Beate Krauβ;K. Decker
嗜烟节杆菌是一种革兰氏阳性需氧土壤细菌，能够以尼古丁作为碳和氮的唯一来源生长。尼古丁分解代谢的初始步骤是由尼古丁脱氢酶、l-和d-特异性6-羟基尼古丁氧化酶和酮脱氢酶催化的。编码这些酶的基因位于160 kb的质粒pAO1上。该质粒的cccDNA纯度高，产率合理。它作为构建质粒噬菌体DNA文库的模板材料。对6-羟基-l-尼古丁氧化酶基因和异三聚体酮脱氢酶亚基基因进行了鉴定、亚克隆和测序。6-hlno基因为1278 bp的开放阅读框；它的管制因素也得到确认。得到的载o-6-羟基-l-尼古丁氧化酶单体的一级结构（46,264.5 Da）与部分氨基酸测序结果一致。6-羟基-l-尼古丁氧化酶和6-羟基-d-尼古丁氧化酶在大肠杆菌中表达，得到高纯度结晶。酮脱氢酶（KDH）为异源三聚体，亚基分子量分别为89,021.71,26,778.65和17,638.88。KDH-A和KDH-B基因并置；KDH-A的停止密码子的A被用在KDH-B的开始密码子上，引起帧移位。KDH-C与KDH-A分离281bp。
Arthrobacter nicotinovorans is a Gram-positive aerobic soil bacterium able to grow on nicotine as its sole source of carbon and nitrogen. The initial steps of nicotine catabolism are catalyzed by nicotine dehydrogenase, the l- and d-specific 6-hydroxynicotine oxidases, and ketone dehydrogenase. The genes encoding these enzymes reside on a 160 kb plasmid, pAO1. The cccDNA of this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a lambda-phage DNA library of the plasmid. The genes coding for 6-hydroxy-l-nicotine oxidase and for the subunits of the heterotrimeric ketone dehydrogenase were identified, subcloned and sequenced. The 6-hlno gene was identified as a 1278 bp open reading frame; its regulatory elements were also recognized. The derived primary structure of the monomer of apo-6-hydroxy-l-nicotine oxidase (46,264.5 Da) agrees with the data obtained by partial amino acid sequencing. 6-Hydroxy-l-nicotine oxidase and 6-hydroxy-d-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized. Ketone dehydrogenase (KDH) was found to be a heterotrimer with subunits of molecular mass 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxtaposed; the A of the stop codon of KDH-A is used in the start codon of KDH-B, eliciting a frame shift. KDH-C is separated from KDH-A by 281 bp.