Cloning, sequencing, and expression of UDP-glucose pyrophosphorylase gene from Acetobacter xylinum BRC5.
A UDP-glucose pyrophosphorylase (UGPase) gene from Acetobacter xylinum BRC5 has been cloned, sequenced, and expressed in Escherichia coli. The gene consists of 867 nucleotides and encodes a polypeptide of 289 amino acid residues with a calculated molecular mass of 31,493 Da. The amino acid sequences of the enzyme showed an 85.8% identity to those of an enzyme from A. xilinum ATCC 23768. A polyhistidine-UGPase fusion enzyme was expressed and purified from the transformed E. coli. The enzyme showed a 35,620-Da single protein band on SDS/PAGE and an about 160,000-Da protein band on 8-16% pore-gradient polyacrylamide gel, indicating the enzyme may be a tetramer or pentamer composed of four or five identical subunits. Kinetic analysis of the enzyme showed a typical Michaelis-Menten substrate saturation pattern, from which Km and Vmax were calculated to be 3.22 mM and 175.4 micromol x min(-1) x mg(-1) for UDP-glucose and 0.24 mM and 69.4 micromol x min(-1) x mg(-1) for PPi, respectively, required Mg2+ for maximal activity, and was inhibited by free pyrophosphate. Computer-aided comparison of the Acetobacter enzyme sequence with those of other bacterial enzymes found significant similarities among them and predicted that Lys84 is a catalytically important residue. Lys84 in the enzyme, which was also conserved in other bacterial enzyme sequences, was replaced by arginine or leucine. The K84R mutant enzyme was successfully expressed in E. coli and showed enzyme activity (63% of the wild-type enzyme activity), but K84L was not isolated in stable form. These results suggest that Lys84 is significant in not only catalysis but also maintenance of the active structure.
Koo
H M
HM
Department of Biochemistry, College of Science, Bioproducts Research Center, Yonsei University, Seoul, Korea.
Yim
S W
SW
Lee
C S
CS
Pyun
Y R
YR
Kim
Y S
YS
D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D041822:Gluconacetobacter xylinus / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D017421:Sequence Analysis; D014169:Transformation, Bacterial; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics* / Q000302:isolation & purification
D005957:UTP-Glucose-1-Phosphate Uridylyltransferase