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

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dc.contributor.authorHosung Sohn-
dc.contributor.authorYong Sam Kim-
dc.contributor.authorU H Jin-
dc.contributor.authorS J Suh-
dc.contributor.authorSang Chul Lee-
dc.contributor.authorDae Sil Lee-
dc.contributor.authorJeong Heon Ko-
dc.contributor.authorC H Kim-
dc.date.accessioned2017-04-19T09:05:40Z-
dc.date.available2017-04-19T09:05:40Z-
dc.date.issued2006-
dc.identifier.issnI0000096-
dc.identifier.uri10.1007/s10719-006-9004-1ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/7710-
dc.description.abstractExpanding 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.-
dc.publisherSpringer-
dc.titleAlteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction-
dc.title.alternativeAlteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction-
dc.typeArticle-
dc.citation.titleGlycoconjugate Journal-
dc.citation.number9-
dc.citation.endPage625-
dc.citation.startPage619-
dc.citation.volume23-
dc.contributor.affiliatedAuthorYong Sam Kim-
dc.contributor.affiliatedAuthorSang Chul Lee-
dc.contributor.affiliatedAuthorJeong Heon Ko-
dc.contributor.alternativeName손호성-
dc.contributor.alternativeName김용삼-
dc.contributor.alternativeName진운호-
dc.contributor.alternativeName서석종-
dc.contributor.alternativeName이상철-
dc.contributor.alternativeName이대실-
dc.contributor.alternativeName고정헌-
dc.contributor.alternativeName김철호-
dc.identifier.bibliographicCitationGlycoconjugate Journal, vol. 23, no. 9, pp. 619-625-
dc.identifier.doi10.1007/s10719-006-9004-1-
dc.subject.keywordADP-glucose pyrophosphorylase (ATP:α-glucose-1-phosphate adenylytransferase, EC 2.7.7.27)-
dc.subject.keyworderror-prone polymerase chain reaction-
dc.subject.keywordthermus caldophilus GK-24-
dc.subject.localADP-glucose pyrophosphorylase (ATP:α-glucose-1-phosphate adenylytransferase, EC 2.7.7.27)-
dc.subject.localerror-prone polymerase chain reaction-
dc.subject.localthermus caldophilus GK-24-
dc.subject.localThermus caldophilus GK24-
dc.description.journalClassY-
Appears in Collections:
Division of Biomedical Research > Genome Editing Research Center > 1. Journal Articles
Division of Biomedical Research > Metabolic Regulation Research Center > 1. Journal Articles
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