CLONING AND NUCLEOTIDE-SEQUENCES OF LUX GENES AND CHARACTERIZATION OF LUCIFERASE OF XENORHABDUS-LUMINESCENS FROM A HUMAN WOUND
CLONING AND NUCLEOTIDE-SEQUENCES OF LUX GENES AND CHARACTERIZATION OF LUCIFERASE OF XENORHABDUS-LUMINESCENS FROM A HUMAN WOUND
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DOI：
10.1128/jb.173.4.1399-1405.1991
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发表时间：
1991-02-01
影响因子：
3.2
通讯作者：
TU, SC
中科院分区：
文献类型：
--
作者：
XI, L;CHO, KW;TU, SC
发光杆菌是唯一已知的从人类(伤口)来源分离的发光细菌。构建了含有荧光素酶α亚基和β亚基的发光菌HW LuxA和LuxB基因，以及LuxC、LuxD和部分LuxE基因的重组表达载体。测定了这些lux基因的核苷酸序列，并在大肠杆菌宿主细胞中实现了荧光素酶基因的高效表达。克隆的荧光素酶与野生型荧光素酶在体外的生物发光动力学性质没有什么区别。与早期的报道相反，我们的发现表明，发光X.Lighescens HW荧光素酶的α亚基(362个氨基酸残基，M(R)41,389)和β亚基(324个氨基酸残基，M(R)37,112)的比活性和大小在已知发光细菌系统中都是不寻常的。观察到发光菌HW荧光素酶的α和β亚基与其他发光细菌的序列有显著的同源性。然而，发光菌HW荧光素酶的不同寻常之处在于4a-羟基过氧黄素中间体的高度稳定性和对醛底物抑制的敏感性。
Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase alpha and beta subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate that neither the specific activity nor the size of the alpha (362 amino acid residues, M(r) 41,389) and beta (324 amino acid residues, M(r) 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the alpha and beta subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.