Molecules and Cells

  • 제45권7호
  • /
  • Pages.495-501
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  • 2022
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  • 1016-8478(pISSN)
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  • 0219-1032(eISSN)
한국분자세포생물학회 (Korean Society for Molecular and Cellular Biology)
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The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa

  • Kim, Seheon (Department of Chemistry, College of Natural Sciences, Soongsil University) ;
  • Koh, Seri (Department of Chemistry, College of Natural Sciences, Soongsil University) ;
  • Kang, Wonchull (Department of Chemistry, College of Natural Sciences, Soongsil University) ;
  • Yang, Jin Kuk (Department of Chemistry, College of Natural Sciences, Soongsil University)
  • 투고 : 2021.10.29
  • 심사 : 2022.02.24
  • 발행 : 2022.07.31
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초록

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD+ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60℃ and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD+) were determined. The crystal structure represents that the subunit has more compact structure than homologs' structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

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과제정보

This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (grant No. 2019R1F1A1063268 to J.K.Y., grant No. 2022R1C1C1004221 to W.K., and grant No. 2021R1A6A1A10044154 to W.K.). We would like to thank the staff at the 11C beamline at Pohang Accelerator Laboratory for support during data collection and the staff at the Korea Basic Science Institute (KBSI) for SEC-MALS and CD measurements.

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