Advertisement

Enhancing thermostability and removing hemin inhibition of Rhodopseudomonas palustris 5-aminolevulinic acid synthase by computer-aided rational design

  • Zijian Tan
  • Jing Zhao
  • Jiuzhou Chen
  • Deming Rao
  • Wenjuan Zhou
  • Ning Chen
  • Ping Zheng
  • Jibin Sun
  • Yanhe Ma
Original Research Paper
  • 27 Downloads

Abstract

Objective

To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy.

Results

Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 μM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively.

Conclusion

H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.

Keywords

5-Aminolevulinic acid synthase Hemin inhibition Rational design Rhodopseudomonas palustris Thermal stability 

Notes

Acknowledgements

We are grateful for the financial support from National Natural Science Foundation of China (No. 21606251), the Key Research Program of the Chinese Academy of Sciences (No. KFZD-SW-212), the Tianjin Municipal City, the first “Special Support Plan for Talents Development” and “High-level Innovation and Entrepreneurship Team”, and Science and Technology Project of Tianjin (Nos. 15PTCYSY00020 and 14ZCZDSY00058). We thank Taiwo Dele-Osibanjo (Tianjin Institute of Industrial Biotechnology) for critical reading and editing of the manuscript.

Supporting information

Supplementary Table 1—Primers used in this study.

Supplementary Table 2—The occurring frequency of amino acids at histidine positions. The data are shown as percentage values.

Supplementary Table 3—The values of the folding free energy change (∆∆G) predicted by FoldX. The unit is kcal/mol.

Supplementary Fig. 1—3D model of wild-type RP-ALAS. The homology model of wild-type RP-ALAS was constructed using the crystal structure of ALAS from R. capsulatus (PDB code: 2BWP) as template.

Supplementary Fig. 2—SDS-PAGE analysis of the purified ALASs. Lane M, protein marker; lane 1, wild-type; lane 2, H29R; lane 3, H15K.

Supplementary Fig. 3—Absorption spectra of hemin-RP-ALAS (black squares), hemin-H29R (green triangles) and hemin-H15K (red circles) complex. The concentrations of hemin were 20 and of ALAS were (a) 10 μM (874 μg/mL), (b) 4 μM (349.6 μg/mL) and (c) 2 μM (174.8 μg/mL), respectively.

Supplementary Fig. 4—ALA production using Corynebacterium glutamicum ATCC 13032 expressing wild-type, H29R and H15K.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2627_MOESM1_ESM.docx (805 kb)
Supplementary material 1 (DOCX 805 kb)

References

  1. Astner I, Schulze JO, van den Heuvel J, Jahn D, Schubert WD, Heinz DW (2005) Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans. EMBO J 24:3166–3177CrossRefGoogle Scholar
  2. Burnham BF (1970) δ-Aminolevulinic acid synthase (Rhodopseudomonas sphaeroides). Methods Enzymol 17:195–204CrossRefGoogle Scholar
  3. Charbonneau DM, Beauregard M (2013) Role of key salt bridges in thermostability of G. thermodenitrificans EstGtA2: distinctive patterns within the new bacterial lipolytic enzyme subfamily XIII.2. PLoS ONE 8:e76675CrossRefGoogle Scholar
  4. Dubay KH, Bothma JP, Geissler PL (2011) Long-range intra-protein communication can be transmitted by correlated side-chain fluctuations alone. PLoS Comput Biol 7:e1002168CrossRefGoogle Scholar
  5. Igarashi J, Murase M, Iizuka A, Pichierri F, Martinkova M, Shimizu T (2008) Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins. J Biol Chem 283:18782–18791CrossRefGoogle Scholar
  6. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780CrossRefGoogle Scholar
  7. Kitatsuji C, Ogura M, Uchida T, Ishimori K, Aono S (2014) Molecular mechanism for heme-mediated inhibition of 5-aminolevulinic acid synthase 1. Bull Chem Soc Jpn 87:997–1004CrossRefGoogle Scholar
  8. Kou F, Zhao J, Liu J, Sun C, Guo Y, Tan Z, Cheng F, Li Z, Zheng P, Sun J (2018) Enhancement of the thermal and alkaline pH stability of Escherichia coli lysine decarboxylase for efficient cadaverine production. Biotechnol Lett 40:719–727CrossRefGoogle Scholar
  9. Kuhl T, Sahoo N, Nikolajski M, Schlott B, Heinemann SH, Imhof D (2011) Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach. ChemBioChem 12:2846–2855CrossRefGoogle Scholar
  10. Kumar A, Wu G, Wu Z, Kumar N, Liu Z (2018) Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis by site directed mutagenesis. Int J Biol Macromol 117:1216–1223CrossRefGoogle Scholar
  11. Lathrop JT, Timko MP (1993) Regulation by heme of mitochondrial protein-transport through a conserved amino-acid motif. Science 259:522–525CrossRefGoogle Scholar
  12. Lehmann M, Pasamontes L, Lassen SF, Wyss M (2000) The consensus concept for thermostability engineering of proteins. Biochim Biophy Acta 1543:408–415CrossRefGoogle Scholar
  13. Lou JW, Zhu L, Wu MB, Yang LR, Lin JP, Cen PL (2014) c. J Zhejiang Univ Sci B 15:491–499CrossRefGoogle Scholar
  14. Matsui D, Nakano S, Dadashipour M, Asano Y (2017) Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity. Sci Rep 7:9558CrossRefGoogle Scholar
  15. Meng Q, Zhang Y, Ma C, Ma H, Zhao X, Chen T (2015) Purification and functional characterization of thermostable 5-aminolevulinic acid synthases. Biotechnol Lett 37:2247–2253CrossRefGoogle Scholar
  16. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19:1639–1662CrossRefGoogle Scholar
  17. Porebski BT, Buckle AM (2016) Consensus protein design. Protein Eng Des Sel 29:245–251CrossRefGoogle Scholar
  18. Rod TH, Radkiewicz JL, Brooks CL III (2003) Correlated motion and the effect of distal mutations in dihydrofolate reductase. Proc Natl Acad Sci USA 100:6980–6985CrossRefGoogle Scholar
  19. Tripp KW, Sternke M, Majumdar A, Barrick D (2017) Creating a homeodomain with high stability and DNA binding affinity by sequence averaging. J Am Chem Soc 139:5051–5060CrossRefGoogle Scholar
  20. Wang W, Malcolm BA (1999) Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis. Biotechniques 26:680–682CrossRefGoogle Scholar
  21. Yi ZL, Pei XQ, Wu ZL (2011) Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum. Bioresour Technol 102:3636–3638CrossRefGoogle Scholar
  22. Zhang R, Zhang J, Guo G, Mao X, Tong W, Zhang Y, Wang DC, Hu Y, Zou Q (2011) Crystal structure of Campylobacter jejuni ChuZ: a split-barrel family heme oxygenase with a novel heme-binding mode. Biochem Biophys Res Commun 415:82–87CrossRefGoogle Scholar
  23. Zhang L, Chen J, Chen N, Sun J, Zheng P, Ma Y (2013) Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production. Biotechnol Lett 35:763–768CrossRefGoogle Scholar
  24. Zou Y, Chen T, Feng L, Zhang S, Xing D, Wang Z (2017) Enhancement of 5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in Corynebacterium glutamicum. Biotechnol Lett 39:1369–1374CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.College of Chemical Engineering and Materials ScienceTianjin University of Science & TechnologyTianjinChina
  2. 2.Key Laboratory of Systems Microbial BiotechnologyChinese Academy of SciencesTianjinChina
  3. 3.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina
  4. 4.College of BiotechnologyTianjin University of Science & TechnologyTianjinChina

Personalised recommendations