Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction.
Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P) + uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones (H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate.
Sohn
Hosung
H
Proteomics System Research Center, Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejon, South Korea.
Kim
Yong-Sam
YS
Jin
Un-Ho
UH
Suh
Seok-Jong
SJ
Lee
Sang Chul
SC
Lee
Dae-Sil
DS
Ko
Jeong Heon
JH
Kim
Cheorl-Ho
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology* / Q000235:genetics; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D004269:DNA, Bacterial; D005958:Glucosephosphates; D014537:Uridine Diphosphate N-Acetylglucosamine; C031590:glucose-1-phosphate; D051599:Glucose-1-Phosphate Adenylyltransferase; D014532:Uridine Diphosphate Glucose