Nitrile pathway involving Acyl-CoA synthetase - Overall metabolic gene organization and purification and characterization of the enzyme
Nitrile pathway involving Acyl-CoA synthetase - Overall metabolic gene organization and purification and characterization of the enzyme
复制标题
DOI：
10.1074/jbc.m405686200
复制
发表时间：
2005-03-11
影响因子：
4.8
通讯作者：
Kobayashi, M
中科院分区：
文献类型：
--
作者：
Hashimoto, Y;Hosaka, H;Kobayashi, M
两个开放的阅读框架（nhpS和阿克萨）被确定为紧接下游的先前描述的绿针假单胞菌B23腈水合酶（NHase）基因簇（编码醛肟脱氢酶，酰胺酶，两个NHase亚基，和一个未表征的蛋白质）。从阿克萨推导的氨基酸序列与酰基辅酶A合成酶（阿克萨）的氨基酸序列相似。阿克萨基因在大肠杆菌中的表达产物对丁酸和CoA具有酰基辅酶A合成酶活性，并可合成丁酰辅酶A。从大肠大肠杆菌中纯化阿克萨，并对其进行了鉴定。重组蛋白的质量通过NH 2-末端氨基酸序列和基质辅助激光解吸电离飞行时间质谱的结果进行验证。丁酸、CoA和ATP的表观Km值分别为0.32 +/- 0.04、0.37 +/- 0.02和0.22 +/- 0.02 mM。阿克萨是一种短链酰基辅酶A合成酶。阿克萨的底物特异性类似于醛肟脱氢酶、腈水合酶和酰胺酶，它们的基因在基因簇中以相同的方向共存。绿针假单胞菌B23在含有丁醛肟作为唯一碳源和氮源的培养基中生长。在所用培养条件下检测醛肟脱氢酶、腈水合酶和酰胺酶的活性以及酰基辅酶A合成酶的活性。此外，在含有丁酸作为唯一碳源的培养基中培养时，还检测到酰基辅酶A合成酶活性。连同邻近的位置的醛肟脱氢酶，腈水合酶，酰胺酶，和酰基辅酶A合成酶基因，这些研究结果表明，这四种酶依次相互关联，在体内利用丁醛肟作为碳和氮源。这是第一次报告的整体“腈途径”（醛肟-->腈-->酰胺-->酸-->酰基辅酶A），包括这些酶。
Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster ( encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 +/- 0.04, 0.37 +/- 0.02, and 0.22 +/- 0.02 mM, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (k(cat)/K-m) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) comprising these enzymes.