Characterization of a putative Pseudomonas UDPglucose pyrophosphorylase.
A UDP-glucose pyrophosphorylase encoding gene was identified through functional complementation screening by using an Escherichia coli galU mutant. Sequence analysis of the gene indicated that it is most likely derived from a Pseud monas sp. The gene is located immediately upstream and transcribed in the same direction of the gor (glutathione reductase) gene and is capable of encoding a protein 30,943 daltons in size. The gene product synthesized in Escherichia coli was purified and its biochemical properties characterized. The recombinant UDP-glucose pyrophosphorylase exhibited a molecular weight of 130 kDa, suggesting a tetrameric organization of the gene product. Two mutant forms of the enzyme were identified. The activity of the mutant enzyme with a tyrosine to histidine (Y26 1H) substitution was found to be greatly reduced. On the other hand, the tyrosine to cysteine (Y84C) substitution resulted in an enzyme that functions normally at 37 degrees C but rather poorly at temperatures lower than 30 degrees C.
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, R. O. C.
H Y
HY
Chang
H C
HC
Huang
J H
JH
Lee
H L
HL
Peng
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000302:isolation & purification*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose