FY2021 Annual Report

Protein Engineering and Evolution Unit
Assistant Professor Paola Laurino

 

Abstract

In the past few decades protein engineering allowed to generating artificial enzymes able to catalize unnatural reactions but also to become importnat tools for synthetic biology. Our Unit is particulary interested in generating new enzymes by directed evolution but also to understand how enzymes evolve in Nature.

1. Staff

  • Dr. Paola Laurino. PI
  • Dr. Madhuri Gade. Researcher
  • Dr. Mirco Dindo. Researcher
  • Dr. Saacnicteh Toledo Patino. Researcher
  • Dr. Benjamin Clifton. Researcher (JSPS Fellow)
  • Dr. Prashant Jain. Researcher
  • Dr. Gen-ichiro Uechi. Technician
  • Mr. Stefano Pascarelli. Graduate Student
  • Mr. Dan Kozome. Graduate Student
  • Mr. Yoshiki Ochiai. Graduate Student
  • Mr. Alessandro Bevilacqua. Graduate Student
  • Ms. Amy Stanton Gooch. Graduate Student
  • Ms. Lilian Magnus. Graduate Student
  • Ms. Mika Uehara. Research Unit Administrator

2. Collaborations

2.1 Topic: Elucidation of Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases

  • Type of collaboration: Joint research
  • Researchers:
    • Professor Colin Jackson, ANU, Australia

2.2 Topic: Enzymatically Active droplets

  • Type of collaboration: Joint research
  • Researchers:
    • Professor Eric R. Dufresne, ETHZ, Switzerland

2.3 Topic: The functional instability of alanine:glyoxylate aminotransferase 

  • Type of collaboration: Joint research
  • Researchers:
    • Professor Barbara Cellini, Perugia University, Italy
    • Professor Giorgio Giardina, Rome University, Italy

3. Activities and Findings

3.1 Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases

Protein conformational changes can facilitate the binding of noncognate substrates and underlying promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here, we analyze human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, noncognate substrates bind in a stable conformation to allow catalysis. In contrast, noncognate substrates sample stable productive binding modes less frequently in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrates likely drove the evolutionary divergence of substrate specificity in these orthologues. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated levels in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.

3.2 Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation 

The tRNA modification m1G37, introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria because it suppresses translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question through experimental evolution of trmD mutant strains of Escherichia coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via duplication or mutation of the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that proS may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

3.3 Sustained enzymatic activity and flow in crowded protein droplets

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

4. Publications

4.1 Journals

  1. Clifton, B. E.; Kozome, D.; Laurino. P.* Efficient exploration of sequence space by sequence-guided protein engineering and design ACS Biochemistry, Perspective, Just accepted https://pubs.acs.org/doi/10.1021/acs.biochem.1c00757
  2.  Gade, M. H.; Lynn Tan, L.; Damry, A. M.; Sandhu, M.; Brock, J. S.; Delanay, A.; Villar-Briones, A.; Jackson, C. J.*; Laurino P.* Substrate dynamics contributes to enzymatic specificity in human and bacterial methionine adenosyltransferases. JACS Au 2021, 1, 12, 2349-2360  https://pubs.acs.org/doi/10.1021/jacsau.1c00464
  3.  Clifton, B. E.; Fariz, M. A.; Uechi, G.; Laurino, P.* Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation. Nucleic Acid Res. 2021, 49, 21, 12467-12485  https://doi.org/10.1093/nar/gkab1067
  4. Testa A.†; Dindo, M.†; Rebane, A.; Nasouri, B.; Style, R.; Golestanian, R.; Dufresne, E.*; Laurino, P.* Sustained Enzymatic activity and Flow in Crowded Protein Droplets  Nat. Comm. 2021, 12, 6293 https://rdcu.be/cAzZP
  5. Chouhan, B. P. S.; Gade, M.; Martinez, D.; Toledo-Patino S.; Laurino P.* Implications of divergence of Methionine AdenosylTransferase in archea. FebsOpenBio 2022, 12,  130-145  https://febs.onlinelibrary.wiley.com/doi/10.1002/2211-5463.13312
  6. Laurino, P.* & Tokuriki N.* Dan Salah Tawfik (1955-2021). Nat. Chem. Biol. 2021, 17, 930.  https://www.nature.com/articles/s41589-021-00864-w

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

  1. Mirco Dindo, "SIB reunion (online)", Apr 2021.
  2. Madhuri Gade, "The 35th Annual Symposium of The Protein Society", Jun 7 - Jun 14. 2021.
  3. Mirco Dindo, "Internal Seminar at OIST", Jun 2021.
  4. Yoshiki Ochiai, “Young meeting of Protein Science Society of Japan(online)", Jun 14. 2021.
  5. Benjamin Clifton, “The 21st Annual Meeting of the Protein Science Society of Japan (online)”, Jun 16 - Jun 18. 2021.
  6. Benjamin Clifton, “The 22nd Annual Meeting of the RNA Society of Japan (online)”, Jul 7- Jul 9. 2021.
  7. Saacnicteh Toledo Patino, “Research Appreciation Month(RAM)", Jul 2021
  8. Saacnicteh Toledo Patino, “Protein Society Symposium Anniversary", Jul 2021
  9. Paola Laurino, “ACA meeting", Jul 30 - Aug 5. 2021.
  10. Saacnicteh Toledo Patino, “Origin Of Life Conference", Aug 2021
  11. Saacnicteh Toledo Patino, “Molecular Origins of Life", Aug 25 - Aug 27. 2021
  12. Mirco Dindo, “Molecular Origins of Life Conference", Aug 25 - Aug 27. 2021.
  13. Mirco Dindo, “SIB conference", Sep 23 - Sep 24. 2021.
  14. Madhuri Gade, “International Conference on Interdisciplinary Approaches in Chemical Sciences- (IACS) MES Abasaheb Garware College(online)", Oct 21-Oct 23. 2021.
  15. Paola Laurino, “The International Conference on interdisciplinary Approches in chemical sciences (IACS-2021)", Oct 22. 2021.
  16. Paola Laurino, “The 27th East Asia Joint Symposium", Oct 28. 2021.
  17. Madhuri Gade, “New Proteins by Evolution and Engineering Mini-symposium(online)", Nov 15- Nov 17. 2021.
  18. Saacnicteh Toledo Patino,“New Proteins by Evolution and Engineering Mini-symposium(online)", Nov 15- Nov 17. 2021.
  19. Paola Laurino, “Minnesota University, seminar series of the biotechnology institute", Dec 3. 2021.
  20. Yoshiki Ochiai, “Pacifichem2021", Dec 19. 2021.
  21. Paola Laurino ELSI (Earth-life science institute) Tokyo Institute of Technology", Jan 28. 2022.
  22. Benjamine Clifton, “OIST internal seminar series", Feb 18. 2022.
  23. Paola Laurino, “The Protein Society Webinar", Mar 9. 2022.

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

6.1 Seminar "The editorial process at Nature Catalysis"

  • Date: May 11, 2021
  • Venue: Zoom
  • Speaker: Dr. Davide Esposito (Chief Editor of Nature Catalysis)

6.2 Seminar "Engineering and evolution of nonviral protein capsids packaging RNA"

  • Date: July 6, 2021
  • Venue: Zoom
  • Speaker: Dr. Naohiro Terasaka (The University of Tokyo Graduate School of Science)

6.3 NPEE Mini-Symposium 2021

  • Date: Nov 15-17, 2021
  • Venue: Zoom
  • Speakers:
    • Dr. Shelley Copley University of Colorado (USA)
    • Dr. Janine Copp The University of British Columbia (Canada)
    • Dr. Nobuyasu Koga Institute for Molecular Science (Japan)
    • Dr. Ruchi Anand Department of Chemistry, IIT Bombay (India)
    • Dr. Sergio Peisajovich Illumina (USA)
    • Dr. Colin Jackson The Australian National University (Australia)
    • Dr. Tomoaki Matsuura Earth-Life Science Institute (ELSI) Tokyo Institute of Technology (Japan)
    • Dr. Klara Hlouchova Charles University (Czech Republic)
    • Dr. Birte Hoecker The University of Bayreuth (Germany)
    • Dr. Vikram Alva Max Planck Institute for Developmental Biology (Germany)

6.4 Seminar " Bridging the genotype-phenotype-fitness divide: from protein interfaces to organismal fitness"

  • Date: Feb 21, 2022
  • Venue: Zoom
  • Speaker: Dr. Shimon Bershtein (Ben-Gurion University of the Negev Department of Life Sciences)

6.5 Seminar "The role of structural dynamics in protein evolvability"

  • Date: Feb 22, 2022
  • Venue: Zoom
  • Speaker: Dr. Yusran Abdillah Muthahari (Forth Institute of Molecular Biology & Biotechnology)

7. Other

Nicteh and Mirco's images were selected for [OIST 10th] OIST Images of Science Campus Exhibition![OIST 10th] OIST Images of Science Campus Exhibition | OIST Groups