Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319
Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319
复制标题
DOI：
10.1007/s00253-006-0355-0
复制
发表时间：
2006-04
影响因子：
5
通讯作者：
Luo Liu;R. Schmid;V. Urlacher
中科院分区：
文献类型：
--
作者：
Luo Liu;R. Schmid;V. Urlacher
在红球菌红球菌株DSM 44319中鉴定出细胞色素P450单加氧酶IV类的新成员。 ruber 尚未测序，使用简并引物扩增了 P450 样基因片段。 P450 样 DNA 片段的侧翼区域通过使用聚合酶链式反应的定向基因组步移进行鉴定。初级蛋白质结构表明是一种天然的自给自足的融合蛋白，由铁氧还蛋白、含黄素还原酶和 P450 单加氧酶组成。酶中发现的唯一黄素是核黄素 5'-单磷酸。该酶在大肠杆菌中成功表达、纯化和表征。在NADPH存在下，P450单加氧酶对多环芳烃萘、茚、苊、甲苯、芴、间二甲苯和乙苯表现出羟基化活性。萘、苊和芴的转化产生各自的环单羟基化代谢物。烷基芳烃如甲苯、间二甲苯和乙苯仅在侧链处羟基化。这种新酶氧化此类化合物的能力使其成为污染物生物降解的潜在候选者和有吸引力的合成生物催化剂。
A new member of class IV of cytochrome P450 monooxygenases was identified inRhodococcus ruberstrain DSM 44319. As the genome ofR. ruberhas not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed inEscherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene,m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene,m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.