Cloning, expression and characterization of glucose-1-phosphate thymidylyltransferase (strmlA) from Thermus caldophilus.
dTDP-L-Rhamnose biosynthetic gene cluster was cloned from Thermus caldophilus. A cluster of four open reading frames, strmlA, B, C and D, responsible for the production of dTDP-L-rhmanose, was screened from the genomic library. Thermophilic glucose-1-phosphate thymidylyltransferase, encoding 356 amino acids with a calculated molecular weight 38 kDa, was expressed under the control of the tac promoter in E. coli. The expressed enzyme, stRmlA is thermostable up to 70 degrees C and apparently retained its activity even up to 90 degrees C. It shares 73% sequence identity to glucose-1-phosphate thymidylyltransferase from Streptomyces argillaceus. Amino acid sequence comparison of stRmlA with ten glucose-1-phosphate thymidylyltransferases indicated higher number of unusual hydrophobic residues (A10, A58, A69, A252, V225, V257, V265, 1242 and 1246) and charged residues (D43, D160, D248, D249, D315, H124, H201, H283 and H354) in stRmlA.
Parajuli
Niranjan
N
Institute of Biomolecule Reconstruction, Sun Moon University, #100, Kalsanri, Tangjeonmyun, Asan-si, Chungnam 336-708, Republic of Korea.
Lee
Dea-Sil
DS
Lee
Hei Chan
HC
Liou
Kwangkyoung
K
Sohng
Jae Kyung
JK
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013824:Thermus / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase