Seibert Volker, Thiel Monika, Hinner Isabelle-S, Schlömann Michael
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany.
Microbiology (Reading). 2004 Feb;150(Pt 2):463-472. doi: 10.1099/mic.0.26602-0.
A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335(T) (DSM 531(T)), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335(T) maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335(T) cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335(T) with this substrate.
从能够利用4-氟苯甲酸作为唯一碳源的真养产碱菌335(T)(DSM 531(T))中克隆出了一个包含马来酰乙酸还原酶(EC 1.3.1.32)基因的基因簇。对该基因簇进行测序表明，真养产碱菌335(T)的马来酰乙酸还原酶基因macA是一个新基因簇的一部分，该基因簇与已知的编码马来酰乙酸还原酶的基因簇无关。该基因簇还包含一个假定的膜转运蛋白基因macB，可能与macA共转录，以及一个假定的调控基因macR，它与macBA反向转录。发现MacA与洋葱伯克霍尔德菌AC1100的马来酰乙酸还原酶TftE关系最为密切(相同位置为62%)，与洋葱伯克霍尔德菌R34二硝基甲苯分解代谢基因簇中的一个假定的马来酰乙酸还原酶关系密切(相同位置为61%)。通过在大肠杆菌中表达macA，证实macA编码一种功能性的马来酰乙酸还原酶。从以4-氟苯甲酸培养的真养产碱菌335(T)细胞中纯化马来酰乙酸还原酶，从而确定了纯化蛋白的N端序列，结果表明该序列与从克隆的macA基因预测的序列相同，从而证明该基因实际上参与了真养产碱菌335(T)利用该底物的生长过程。
