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Crystal structure of Clostridium thermocellum ribose-5-phosphate isomerase B reveals properties critical for fast enzyme kinetics

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Abstract

Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of d-ribose 5-phosphate (R5P) to d-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, d-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-Å resolution or higher, of CtRpi alone and bound to its substrates, R5P, d-ribose, and d-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi.

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Acknowledgments

We are grateful to Dr. Y. G. Kim and Dr. K. J. Kim for their assistance at the beamline 4A and 6C of Pohang Light Source (PLS) in South Korea, and to staff members at the beamline 17A of the photon factory (KEK), Japan. This work was supported by a WCU (World Class University, R33-2008-000-1071) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology, South Korea; by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0011666); and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST, No. 2010-0027545).

Accession numbers

Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 3HE8 (Apo-CtRpi), 3HEE (CtRpi-R5P), 3PH3 (CtRpi-RBO), and 3PH4 (CtRpi-ALO).

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Authors and Affiliations

  1. Department of Advanced Technology Fusion, Konkuk University, Hwayang dong, Gwangjin-gu, Seoul, 143-701, South Korea

    Junho Jung, Jin-Kwang Kim & Lin-Woo Kang

  2. Department of Bioscience and Biotechnology, Konkuk University, Hwayang dong, Gwangjin-gu, Seoul, 143-701, South Korea

    Soo-Jin Yeom & Deok-Kun Oh

  3. Department of Green Life Science, College of Convergence, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul, 110-743, South Korea

    Yeh-Jin Ahn

Authors
  1. Junho Jung
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  2. Jin-Kwang Kim
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  3. Soo-Jin Yeom
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  4. Yeh-Jin Ahn
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  5. Deok-Kun Oh
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  6. Lin-Woo Kang
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Corresponding authors

Correspondence to Deok-Kun Oh or Lin-Woo Kang.

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Junho Jung and Jin-Kwang Kim equally contributed to this paper.

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Jung, J., Kim, JK., Yeom, SJ. et al. Crystal structure of Clostridium thermocellum ribose-5-phosphate isomerase B reveals properties critical for fast enzyme kinetics. Appl Microbiol Biotechnol 90, 517–527 (2011). https://doi.org/10.1007/s00253-011-3095-8

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  • DOI: https://doi.org/10.1007/s00253-011-3095-8

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