Protein of unknown function from variovorax paradoxus with amino acid substitution n174k is able to form schiff base with plp molecule

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Pyridoxal-5'-phosphate (PLP)-dependent enzymes are one of the most widely represented groups of enzymes in organisms, performing more than 150 different catalytic functions. Based on the three-dimensional structure, members of this group are divided into seven (I-VII) different fold types. Cofactor binding in these enzymes occurs due to the formation of a Schiff base with a conserved lysine residue located in the active site. A recently discovered protein from the bacterium Variovorax paradoxus (VAPA), which belongs to the IV fold type and has significant structural similarity to transaminases, contains an asparagine residue at the catalytic lysine position in the transaminases and, as a result, cannot form a Schiff base with PLP and does not have aminotransferase activity. In this research, a point mutant of VAPA protein with the N174K substitution was obtained and its 3D structure was determined. Analysis of the structural data showed that the introduced mutation restores the ability of VAPAN174K to form a Schiff base with a cofactor.

Full Text

Restricted Access

About the authors

I. O. Ilyasov

Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Author for correspondence.
Email: kmb@inbi.ras.ru
Russian Federation, Moscow

M. E. Minyaev

Institute of Organic Chemistry, Russian Academy of Sciences

Email: kmb@inbi.ras.ru
Russian Federation, Moscow

T. V. Rakitina

National Research Centre “Kurchatov Institute”

Email: kmb@inbi.ras.ru
Russian Federation, Moscow

A. K. Bakunova

Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: kmb@inbi.ras.ru
Russian Federation, Moscow

V. O. Popov

Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: kmb@inbi.ras.ru
Russian Federation, Moscow

E. Y. Bezsudnova

Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: kmb@inbi.ras.ru
Russian Federation, Moscow

K. M. Boyko

Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: kmb@inbi.ras.ru
Russian Federation, Moscow

References

  1. Boyko K.M., Matyuta I.O., Nikolaeva A.Y. et al. // Crystals. 2022. V. 12. P. 619. https://doi.org/10.3390/cryst12050619
  2. Christen P., Mehta P.K. // Chem. Rec. 2001. V. 1. P. 436. https://doi.org/10.1002/tcr.10005
  3. Bezsudnova E.Y., Popov V.O., Boyko K.M. // Appl. Microbiol. Biotechnol. 2020. V. 104. P. 2343. https://doi.org/10.1007/s00253-020-10369-6
  4. Catazaro J., Caprez A., Guru A. et al. // Proteins. 2014. V. 82. P. 2597. https://doi.org/10.1002/prot.24624
  5. Bezsudnova E.Y., Boyko K.M., Popov V.O. // Biochemistry (Moscow). 2017. V. 82. P. 1572. https://doi.org/10.1134/S0006297917130028
  6. Bezsudnova E.Y., Dibrova D.V., Nikolaeva A.Y. et al. // J. Biotechnol. 2018. V. 271. P. 26. https://doi.org/10.1016/j.jbiotec.2018.02.005
  7. Liang J., Han Q., Tan Y. et al. // Front. Mol. Biosci. 2019. V. 6. P. 4. https://doi.org/10.3389/fmolb.2019.00004
  8. Cook P.D., Thoden J.B., Holden H.M. // Protein Sci. 2006. V. 15. P. 2093. https://doi.org/10.1110/ps.062328306
  9. Evans P.R., Murshudov G.N. // Acta Cryst. D. 2013. V. 69. P. 1204. https://doi.org/10.1107/S0907444913000061
  10. Vagin A., Teplyakov A. // J. Appl. Cryst. 1997. V. 30. P. 1022. https://doi.org/10.1107/S0021889897006766
  11. Collaborative Computational Project // Acta Cryst. D. 1994. V. 50. P. 760. https://doi.org/10.1107/S0907444994003112
  12. Murshudov G.N., Skubak P., Lebedev A.A. et al. // Acta Cryst. D. 2011. V. 67. P. 355. https://doi.org/10.1107/S0907444911001314
  13. Emsley P., Cowtan K. // Acta Cryst. D. 2004. V. 60. P. 2126. https://doi.org/10.1107/S0907444904019158
  14. Wallace A.C., Laskowski R.A., Thornton J.M. // Protein Eng. Des. Sel. 1995. V. 8. P. 127. https://doi.org/10.1093/protein/8.2.127
  15. Dai Y.N., Chi C.B., Zhou K. et al. // J. Biol. Chem. 2013. V. 288. P. 22985. https://doi.org/10.1074/jbc.M113.480335

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Dimer structure of the VAPAN174K variant. The N-terminal domain is shown in green, the C-terminal domain in red, the interdomain loop in light green, and the adjacent subunit in gray. The PLP molecule covalently linked to the K174 residue is shown in yellow for one of the subunits. The inset at the top right shows a photograph of the VAPAN174K variant crystal used in the X-ray crystallography.

Download (507KB)
Indexing metadata
3. Fig. 2. Structural differences between the VAPAN174K variant (color scheme similar to Fig. 1) and VAPAwt (translucent gray, PDB ID: 7Z79) in the putative active site. Electron density (2Fo–Fc map) for the internal aldimine is shown as a grid surface (cutoff level 1σ), dotted lines indicate regions that differ in conformation from those in VAPAwt. Additional color codes: blue – O-pocket loop formed by residues of the adjacent subunit; orange – βX- and βY-strands of the N-terminal domain; black – β-turn of the C-terminal domain; yellow – CNST sequence, which is a loop with a GAGE ​​motif in other transaminases [1].

Download (286KB)
Indexing metadata
4. Fig. 3. Amino acid residues involved in PLP binding in the VAPAN174K variant structure: a – spatial arrangement of residues, color scheme similar to Fig. 2. Solvent molecules are shown as red spheres. For comparison, the position of the PLP molecule in the VAPAwt structure is shown in gray translucent color; b – PLP molecule binding scheme made using LigPlot [15]. Hydrogen bonds are shown as a green dotted line with the corresponding distances in angstroms indicated.

Download (231KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences