Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction |
| |
Authors: | Hosung Sohn Yong-Sam Kim Un-Ho Jin Seok-Jong Suh Sang Chul Lee Dae-Sil Lee Jeong Heon Ko Cheorl-Ho Kim |
| |
Institution: | (1) Proteomics System Research Center, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Daejon, 305-333, South Korea;(2) Molecular and Cellular Glycobiology Unit, Department of Biological Sciences, Sungkyunkwan University, Chunchun-Dong 300, Jangan-Gu, Suwon, Kyunggi-Do, 440-746, South Korea |
| |
Abstract: | 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.
The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study. |
| |
Keywords: | ADP-glucose pyrophosphorylase (ATP:α -glucose-1-phosphate adenylytransferase EC 2 7 7 27) Thermus caldophilus GK-24 Error-prone polymerase chain reaction |
本文献已被 PubMed SpringerLink 等数据库收录! |
|