Substrate inactivation of bacterial l-aspartate α-decarboxylase from Corynebacterium jeikeium K411 and improvement of molecular stability by saturation mutagenesis

Abstract

Bacterial l-aspartate α-decarboxylase (PanD) is a potential biocatalyst for the green production of β-alanine, an important block chemical for manufacturing nitrogen-containing chemicals in bio-refinery field. It was reported that the poor catalytic stability caused by substrate inactivation limited the large-scale application. Here, we investigated the characters of inactivation by l-aspartate of PanD from Corynebacterium jeikeium (PDCjei), and found that l-aspartate induced a time-, and concentration-dependent inactivation of PDCjei with the values of KI and kinact being 288.4 mM and 0.235/min, respectively. To improve the catalytic stability of PDCjei, conserved amino acid residues essential to catalytic stability were analyzed by comparing the discrepancy in the observed inactivation rate of various sources. By an efficient colorimetric high-throughput screening method, four mutants with 3.18–24.69% higher activity were obtained from mutant libraries. Among them, the best mutation (R3K) also performed 66.38% higher catalytic stability than the wild type, showing great potential for industrial bio-production of β-alanine.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Anton DL, Kutny R (1987) Mechanism of substrate inactivation of Escherichia coli S-adenosylmethionine decarboxylase. Biochemistry 26:6444–6447. https://doi.org/10.1021/bi00394a022

    CAS  Article  PubMed  Google Scholar 

  2. Blancquaert L, Baba SP, Kwiatkowski S, Stautemas J, Stegen S, Barbaresi S, Chung W, Boakye AA, Hoetker JD, Bhatnagar A, Delanghe J, Vanheel B, Veiga-da-Cunha M, Derave W, Everaert I (2016) Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by β-alanine transamination. J Physiol 594:4849–4863. https://doi.org/10.1113/JP272050

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Chen X, Li Y, Gu Z, Ding Z, Zhang L, Shi G (2017) Expression and characterization of two l-aspartate alpha-decarboxylases. Microbiol China 44:2337–2344. https://doi.org/10.13344/j.microbiol.china.170009

    Article  Google Scholar 

  4. Cronan JE (1980) Beta-alanine synthesis in Escherichia coli. J Bacteriol 141:1291–1297

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Cui W, Shi Z, Fang Y, Zhou L, Ding N, Zhou Z (2014) Significance of Arg3, Arg54, and Tyr58 of l-aspartate alpha-decarboxylase from Corynebacterium glutamicum in the process of self-cleavage. Biotechnol Lett 36:121–126. https://doi.org/10.1007/s10529-013-1337-9

    CAS  Article  PubMed  Google Scholar 

  6. Dusch N, Puhler A, Kalinowski J (1999) Expression of the Corynebacterium glutamicum panD gene encoding l-aspartate-alpha-decarboxylase leads to pantothenate overproduction in Escherichia coli. Appl Environ Microbiol 65:1530–1539

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Fouad WM, Rathinasabapathi B (2006) Expression of bacterial l-aspartate-alpha-decarboxylase in tobacco increases beta-alanine and pantothenate levels and improves thermotolerance. Plant Mol Biol 60:495–505. https://doi.org/10.1007/s11103-005-4844-9

    CAS  Article  PubMed  Google Scholar 

  8. Khan MU, Baseer MA, Kumar SR, Saravanakumar M, Prasannanjali AG, Gupta PB, Kaushik VK, Handa VK, Islam A (2010) Synthesis and characterization of metabolites and potential impurities of balsalazide disodium, an anti-inflammatory drug. Synth Commun 40:2241–2253. https://doi.org/10.1080/00397910903221068

    CAS  Article  Google Scholar 

  9. Konst PM, Franssen MCR, Scott EL, Sanders JPM (2009) A study on the applicability of l-aspartate alpha-decarboxylase in the bio-based production of nitrogen containing chemicals. Green Chem 11:1646–1652. https://doi.org/10.1039/b902731a

    CAS  Article  Google Scholar 

  10. Lee B, Suh SW (2004) Crystal structure of the Schiff base intermediate prior to decarboxylation in the catalytic cycle of aspartate alpha-decarboxylase. J Mol Biol 340:1–7. https://doi.org/10.1016/j.jmb.2004.04.049

    CAS  Article  PubMed  Google Scholar 

  11. Liu C, Ding Y, Xian M, Liu M, Liu H, Ma Q, Zhao G (2017) Malonyl-CoA pathway: a promising route for 3-hydroxypropionate biosynthesis. Crit Rev Biotechnol 37:933–941. https://doi.org/10.1080/07388551.2016.1272093

    CAS  Article  PubMed  Google Scholar 

  12. Mo Q, Li Y, Wang J, Shi G (2018) Identification of mutations restricting autocatalytic activation of bacterial l-aspartate α-decarboxylase. Amino Acids 50:1433–1440. https://doi.org/10.1007/s00726-018-2620-9

    CAS  Article  PubMed  Google Scholar 

  13. Pei W, Zhang J, Deng S, Tigu F, Li Y, Li Q, Cai Z, Li Y (2017) Molecular engineering of l-aspartate-α-decarboxylase for improved activity and catalytic stability. Appl Microbiol Biotechnol 101:6015–6021. https://doi.org/10.1007/s00253-017-8337-y

    CAS  Article  PubMed  Google Scholar 

  14. Rosario M, Domínguez dMP G, OL JSAJ (2011) A high-throughput screening assay for amino acid decarboxylase activity. Adv Synth Catal 353:2369–2376. https://doi.org/10.1002/adsc.201100386

    CAS  Article  Google Scholar 

  15. Schmitzberger F, Kilkenny ML, Lobley CMC, Webb ME, Vinkovic M, Matak-Vinkovic D, Witty M, Chirgadze DY, Smith AG, Abell C, Blundell TL (2003) Structural constraints on protein self-processing in l-aspartate-alpha-decarboxylase. EMBO J 22:6193–6204. https://doi.org/10.1093/emboj/cdg575

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Shen Y, Zhao L, Li Y, Zhang L, Shi G (2014) Synthesis of beta-alanine from L-aspartate using l-aspartate-alpha-decarboxylase from Corynebacterium glutamicum. Biotechnol Lett 36:1681–1686. https://doi.org/10.1007/s10529-014-1527-0

    CAS  Article  PubMed  Google Scholar 

  17. Smith RC (1988) The structure and molecular characterization of l-aspartate-alpha-decarboxylase from Escherichia coli. Dissertation, Massachusetts Institute of Technology

  18. Song CW, Lee J, Ko YS, Lee SY (2015) Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid. Metab Eng 30:121–129. https://doi.org/10.1016/j.ymben.2015.05.005

    CAS  Article  PubMed  Google Scholar 

  19. Stuecker TN, Bramhacharya S, Hodge-Hanson KM, Suen G, Escalante-Semerena JC (2015) Phylogenetic and amino acid conservation analyses of bacterial l-aspartate-alpha-decarboxylase and of its zymogen-maturation protein reveal a putative interaction domain. BMC Res Notes 8:354–354. https://doi.org/10.1186/s13104-015-1314-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Tauch A, Kaiser O, Hain T, Goesmann A, Weisshaar B, Albersmeier A, Bekel T, Bischoff N, Brune I, Chakraborty T, Kalinowski J, Meyer F, Rupp O, Schneiker S, Viehoever P, Pühler A (2005) Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora. J Bacteriol 187:4671–4682. https://doi.org/10.1128/jb.187.13.4671-4682.2005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Tigu F, Zhang J, Liu G, Cai Z, Li Y (2018) A highly active pantothenate synthetase from Corynebacterium glutamicum enables the production of d-pantothenic acid with high productivity. Appl Microbiol Biotechnol 102:6039–6046. https://doi.org/10.1007/s00253-018-9017-2

    CAS  Article  Google Scholar 

  22. Webb ME, Smith AG, Abell C (2004) Biosynthesis of pantothenate. Nat Prod Rep 21:695–721. https://doi.org/10.1039/b316419p

    CAS  Article  PubMed  Google Scholar 

  23. Williamson JM, Brown GM (1979) Purification and properties of l-aspartate-alpha-decarboxylase, an enzyme that catalyzes the formation of beta-alanine in Escherichia coli. J Biol Chem 254:8074–8082

    CAS  PubMed  Google Scholar 

  24. Yu K, Hu S, Huang J, Mei L-H (2011) A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase. Enzyme Microb Technol 49:272–276. https://doi.org/10.1016/j.enzmictec.2011.06.007

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Science and Technology Major Project (2016YFD0401404), the National Natural Foundation of China (31571817), national first-class discipline program of Light Industry Technology and Engineering (LITE2018-22) and Science and Technology Support Program of Jiangsu province (BE2016628).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Guiyang Shi.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mo, Q., Mao, A., Li, Y. et al. Substrate inactivation of bacterial l-aspartate α-decarboxylase from Corynebacterium jeikeium K411 and improvement of molecular stability by saturation mutagenesis. World J Microbiol Biotechnol 35, 62 (2019). https://doi.org/10.1007/s11274-019-2629-6

Download citation

Keywords

  • l-Aspartate α-decarboxylase
  • β-Alanine
  • Substrate inactivation
  • Catalytic stability
  • Bio-production of β-alanine
  • Saturation mutagenesis