Biochemical characterization of AtuD from Pseudomonas aeruginosa, the first member of a new subgroup of acyl-CoA dehydrogenases with specificity for citronellyl-CoA
Biochemical characterization of AtuD from Pseudomonas aeruginosa, the first member of a new subgroup of acyl-CoA dehydrogenases with specificity for citronellyl-CoA
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DOI：
10.1099/mic.0.2007/014530-0
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发表时间：
2008-03-01
期刊：
影响因子：
2.8
通讯作者：
Jendrossek, Dieter
中科院分区：
文献类型：
--
作者：
Foerster-Frommne, Karin;Chattopadhyay, Ava;Jendrossek, Dieter
atuRABCDEFGH基因簇对铜绿假单胞菌的非环萜利用（Atu）是必需的。大多数Atu蛋白的生物化学功能尚未得到实验验证; AtuC/AtuF是例外，其构成Atu途径的关键酶香叶酰辅酶A羧化酶的两个亚基。在这项研究中，我们研究了AtuD和PA 1535基因产物的生化功能，PA 1535基因产物是一种与AtuD氨基酸序列相关的蛋白质。2D凝胶电泳显示AtuD和PA 1535蛋白在无环萜烯生长的细胞中特异性表达，但在异戊酸或琥珀酸生长的细胞中不存在。突变体分析表明，AtuD，而不是PA 1535的产品是必不可少的非环萜利用。AtuD和PA 1535基因产物在重组大肠杆菌中表达并纯化至均一。纯化的AtuD显示出香茅酰辅酶A脱氢酶活性（V-max 850 mU mg（-1））和对香茅酰辅酶A的高亲和力（K-m 1.6 μ M）。AtuD与辛酰辅酶A、5-甲基己-4-烯酰辅酶A或异戊酰辅酶A无活性。纯化的PA 1535基因产物显示出高的香茅酰辅酶A脱氢酶活性（V-max 2450 mU mg-1），但对香茅酰辅酶A的亲和力显著低于AtuD（K. 18 μ M）。纯化的PA 1535蛋白另外利用辛酰-CoA作为底物（V-max，610 mU mg（-1）; K-m 130 μ M）。据我们所知，AtulD是第一个对萜类分子结构具有底物特异性的酰基辅酶A脱氢酶，并且对功能性Atu途径至关重要。在香茅假单胞菌、水油海蛞蝓和济州海蛞蝓的基因组中发现了潜在的其他类萜-CoA脱氢酶，但在非无环类萜利用细菌中不存在。
The atuRABCDEFGH gene cluster is essential for acyclic terpene utilization (Atu) in Pseudomonas aeruginosa. The biochemical functions of most Atu proteins have not been experimentally verified; exceptions are AtuC/AtuF, which constitute the two subunits of geranyl-CoA carboxylase, the key enzyme of the Atu pathway. In this study we investigated the biochemical function of AtuD and of the PA1535 gene product, a protein related to AtuD in amino acid sequence. 2D gel electrophoresis showed that AtuD and the PA1535 protein were specifically expressed in cells grown on acyclic terpenes but were absent in isovalerate- or succinate-grown cells. Mutant analysis indicated that AtuD but not the product of PA1535 is essential for acyclic terpene utilization. AtuD and PA1535 gene product were expressed in recombinant Escherichia coli and purified to homogeneity. Purified AtuD showed citronellyl-CoA dehydrogenase activity (V-max 850 mU mg(-1)) and high affinity to citronellyl-CoA (K-m 1.6 mu M). AtuD was inactive with octanoyl-CoA, 5-methylhex-4-enoyl-CoA or isovaleryl-CoA. Purified PA1535 gene product revealed high citronellyl-CoA dehydrogenase activity V-max 2450 mU mg-1) but had significantly lower affinity than AtuD to citronellyl-CoA (K. 18 mu M). Purified PA1535 protein additionally utilized octanoyl-CoA as substrate (V-max, 610 mU mg(-1); K-m 130 mu M). To our knowledge AtulD is the first acyl-CoA dehydrogenase with a documented substrate specificity for terpenoid molecule structure and is essential for a functional Atu pathway. Potential other terpenoid-CoA dehydrogenases were found in the genomes of Pseudomonas citronellolis, Marinobacter aquaeolei and Hahella chejuensis but were absent in non-acyclic terpene-utilizing bacteria.