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

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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.

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Acknowledgments

We thank Mr. Dooil Kim for his excellent technical assistance. This work was supported, in part, by a Grant for KRIBB (KGS1120311).

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Authors and Affiliations

  1. Proteomics System Research Center, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Daejon, 305-333, South Korea

    Hosung Sohn, Yong-Sam Kim, Sang Chul Lee, Dae-Sil Lee & Jeong Heon Ko

  2. Molecular and Cellular Glycobiology Unit, Department of Biological Sciences, Sungkyunkwan University, Chunchun-Dong 300, Jangan-Gu, Suwon, Kyunggi-Do, 440-746, South Korea

    Un-Ho Jin, Seok-Jong Suh & Cheorl-Ho Kim

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  1. Hosung Sohn
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  2. Yong-Sam Kim
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  5. Sang Chul Lee
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  6. Dae-Sil Lee
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Correspondence to Jeong Heon Ko or Cheorl-Ho Kim.

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The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study.

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Sohn, H., Kim, YS., Jin, UH. et al. Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction. Glycoconj J 23, 619–625 (2006). https://doi.org/10.1007/s10719-006-9004-1

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  • DOI: https://doi.org/10.1007/s10719-006-9004-1

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