Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria
Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria
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DOI：
10.1128/jb.184.6.1733-1742.2002
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发表时间：
2002-03-01
影响因子：
3.2
通讯作者：
van Beilen, JB
中科院分区：
文献类型：
--
作者：
Smits, THM;Balada, SB;van Beilen, JB
我们从铜绿假单胞菌PAO1、荧光假单胞菌CHA0、博库库假单胞菌AP1、结核分枝杆菌H37Rv和rugosa Prauserella NRRL B-2295中克隆了恶臭假单胞菌GPo1烷烃羟化酶的同源物。序列比较表明，同源物之间的蛋白质序列同源性水平低至35%，假单胞菌烷烃羟化酶之间的亲缘关系与其余烷烃羟化酶一样远。基于观察到GPo1烷烃羟化酶体系中的一个电子转移成分rubbredoxin可以被其他烷烃羟化酶体系中的rubbredoxin所取代，我们开发了3株重组宿主菌株，用于分析新的烷烃羟化酶基因的功能。两个宿主大肠杆菌GEc137和恶臭杆菌GPo12携带了pGEc47DeltaB，该基因编码中链长度烷烃（C-6至C-12）上生长所需的所有蛋白质，除了功能性烷烃羟化酶。第三个宿主是荧光假单胞菌CHA0的alkB敲除衍生物，它不再能够在C-12到C-16烷烃上生长。除了不动杆菌sp. ADP1 AlkM外，所有的烷烃羟化酶同源物都允许三种宿主中的至少一种在正烷烃上生长。
We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47DeltaB, which encodes all proteins necessary for growth on medium-chain-length alkanes (C-6 to C-12), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C-12 to C-16 alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.