Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens.
A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.
Lee
Jung-Kul
JK
BioNgene Company, Ltd., 10-1, 1Ka, Myungryun-Dong, Jongro-Ku, Seoul, 110-521, Korea.
Her
Gun
G
Kim
Sang-Yong
SY
Seo
Jin-Ho
JH
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics*; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D017386:Sequence Homology, Amino Acid
D019883:Alkyl and Aryl Transferases; C039821:decaprenyl pyrophosphate synthetase