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Title 

Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction

Authors 

Hosung SohnYong Sam KimU H JinS J SuhSang Chul LeeDae Sil LeeJeong Heon KoC H Kim

Publisher 

Springer Verlag (Germany)

Issue Date 

2006

Citation 

Glycoconjugate Journal, vol. 23, no. 9, pp. 619-625

Keywords 

ADP-glucose pyrophosphorylase (ATP:α-glucose-1-phosphate adenylytransferase, EC 2.7.7.27)error-prone polymerase chain reactionthermus caldophilus GK-24

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.

ISSN 

0282-0080

Link 

http://dx.doi.org/10.1007/s10719-006-9004-1

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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