Expression in Escherichia coli of UDP-glucose pyrophosphorylase cDNA from potato tuber and functional assessment of the five lysyl residues located at the substrate-binding site.
The entire structural gene for potato tuber UDP-glucose pyrophosphorylase has been amplified from its cDNA by the polymerase chain reaction and inserted into the expression plasmid pTV118-N downstream from the lac promoter. Escherichia coli JM105 cells carrying thus constructed plasmid produced the enzyme to a level of about 5% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The recombinant enzyme purified to homogeneity in two column chromatographic steps was structurally and catalytically identical with the enzyme purified from potato tuber except for the absence of an N-terminal-blocking acetyl group. To examine functional roles of the five lysyl residues that had been identified by affinity labeling studies to be located at or near the active site of the enzyme [Kazuta, Y., Omura, Y., Tagaya, M., Nakano, K., & Fukui, T. (1991) Biochemistry (preceding paper in this issue)], they were replaced individually by glutamine via site-directed mutagenesis. The Lys-367----Gln mutant enzyme was almost completely inactive, and the Lys-263----Gln mutant enzyme had significantly decreased Vmax values with perturbed Km values for pyrophosphate and alpha-D-glucose 1-phosphate. Lys-329----Gln also exhibited increased Km values for these substrates but exhibited Vmax values similar to those of the wild-type enzyme. The two mutant enzymes Lys-409----Gln and Lys-410----Gln showed catalytic properties almost identical with those of the wild-type enzyme. Thus, among the five lysyl residues, Lys-367 is essential for catalytic activity of the enzyme and Lys-263 and Lys-329 may participate in binding of pyrophosphate and/or alpha-D-glucose 1-phosphate.
Katsube
T
T
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
Kazuta
Y
Y
Tanizawa
K
K
Fukui
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D017343:Genes, Plant; D005822:Genetic Vectors; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D004247:DNA; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D008239:Lysine