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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)
  • Received : 2021.10.29
  • Accepted : 2022.02.24
  • Published : 2022.07.31

Abstract

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.

Keywords

Acknowledgement

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.

References

  1. Abrahamson, M.J., Vazquez-Figueroa, E., Woodall, N.B., Moore, J.C., and Bommarius, A.S. (2012). Development of an amine dehydrogenase for synthesis of chiral amines. Angew. Chem. Int. Ed. Engl. 51, 3969-3972. https://doi.org/10.1002/anie.201107813
  2. Baker, P.J., Turnbull, A.P., Sedelnikova, S.E., Stillman, T.J., and Rice, D.W. (1995). A role for quaternary structure in the substrate specificity of leucine dehydrogenase. Structure 3, 693-705. https://doi.org/10.1016/S0969-2126(01)00204-0
  3. Beckett, P.R., Hardin, D.S., Davis, T.A., Nguyen, H.V., Wray-Cahen, D., and Copeland, K.C. (1996). Spectrophometric assay for measuring branched-chain amino acid concentrations: application for measuring the sensitivity of protein metabolism to insulin. Anal. Biochem. 240, 48-53. https://doi.org/10.1006/abio.1996.0329
  4. Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486-501. https://doi.org/10.1107/S0907444910007493
  5. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., and Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook, J.M. Walker, eds. (Totowa, USA: Humana Press), pp. 571-607.
  6. Gu, K.F. and Chang, T.M. (1990a). Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling. Biotechnol. Appl. Biochem. 12, 227-236.
  7. Gu, K.F. and Chang, T.M. (1990b). Production of essential L-branchedchain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+. Biotechnol. Bioeng. 36, 263-269. https://doi.org/10.1002/bit.260360308
  8. Liebschner, D., Afonine, P.V., Baker, M.L., Bunkoczi, G., Chen, V.B., Croll, T.I., Hintze, B., Hung, L.W., Jain, S., McCoy, A.J., et al. (2019). Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861-877. https://doi.org/10.1107/S2059798319011471
  9. Meng, X., Yang, L., Liu, Y., Wang, H., Shen, Y., and Wei, D. (2021). Identification and rational engineering of a high substrate-tolerant leucine dehydrogenase effective for the synthesis of L-tert-leucine. ChemCatChem 13, 3340-3349. https://doi.org/10.1002/cctc.202100414
  10. Ohshima, T., Misono, H., and Soda, K. (1978). Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. J. Biol. Chem. 253, 5719-5725. https://doi.org/10.1016/S0021-9258(17)30327-7
  11. Ohshima, T., Nagata, S., and Soda, K. (1985). Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus. Arch. Microbiol. 141, 407-411. https://doi.org/10.1007/BF00428857
  12. Suzuki, K., Suzuki, K., Koizumi, K., Ichimura, H., Oka, S., Takada, H., and Kuwayama, H. (2008). Measurement of serum branched-chain amino acids to tyrosine ratio level is useful in a prediction of a change of serum albumin level in chronic liver disease. Hepatol. Res. 38, 267-272. https://doi.org/10.1111/j.1872-034X.2007.00268.x
  13. Tajiri, K. and Shimizu, Y. (2013). Branched-chain amino acids in liver diseases. World J. Gastroenterol. 19, 7620-7629. https://doi.org/10.3748/wjg.v19.i43.7620
  14. Turnbull, A.P., Ashford, S.R., Baker, P.J., Rice, D.W., Rodgers, F.H., Stillman, T.J., and Hanson, R.L. (1994). Crystallization and quaternary structure analysis of the NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus. J. Mol. Biol. 236, 663-665. https://doi.org/10.1006/jmbi.1994.1176
  15. Winn, M.D., Ballard, C.C., Cowtan, K.D., Dodson, E.J., Emsley, P., Evans, P.R., Keegan, R.M., Krissinel, E.B., Leslie, A.G., McCoy, A., et al. (2011). Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235-242. https://doi.org/10.1107/S0907444910045749
  16. Yamaguchi, H., Kamegawa, A., Nakata, K., Kashiwagi, T., Fujiyoshi, Y., Tani, K., and Mizukoshi, T. (2021). Leucine dehydrogenase: structure and thermostability. Subcell. Biochem. 96, 355-372. https://doi.org/10.1007/978-3-030-58971-4_10
  17. Yamaguchi, H., Kamegawa, A., Nakata, K., Kashiwagi, T., Mizukoshi, T., Fujiyoshi, Y., and Tani, K. (2019). Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy. J. Struct. Biol. 205, 11-21. https://doi.org/10.1016/j.jsb.2018.12.001
  18. Zhao, Y., Wakamatsu, T., Doi, K., Sakuraba, H., and Ohshima, T. (2012). A psychrophilic leucine dehydrogenase from Sporosarcina psychrophila: purification, characterization, gene sequencing and crystal structure analysis. J. Mol. Catal. B Enzym. 83, 65-72. https://doi.org/10.1016/j.molcatb.2012.06.018