Abstract
In the past few decades, protein engineering allowed the generation of artificial enzymes to catalyze unnatural reactions and become important tools for synthetic biology. Our Unit is particularly interested in generating new enzymes by directed evolution and understanding how new protein functions emerge in Nature.
1. Staff
• Prof. Paola Laurino, Associate Professor
• Dr. Benjamin Clifton, Staff Scientist
• Dr. Samuel Hauf, Staff Scientist
• Dr. Prashant Jain, Postdoctoral Scholar
• Dr. Yusran Abdillah Muthahari, Postdoctoral Scholar
• Dr. Paul Matthay, Postdoctoral Scholar (Jul 2023-)
• Dr. Elsie Dunkley, JSPS Fellow (Feb 2024-)
• Dr. Gen-ichiro Uechi, Technician
• Dr. Mirco Dindo, Visiting Researcher
• Mr. Dan Kozome, PhD Student
• Mr. Yoshiki Ochiai, PhD Student
• Mr. Alessandro Bevilacqua, PhD Student
• Ms. Amy Stanton, Gooch. PhD Student
• Ms. Lilian Magnus, PhD Student
• Ms. Erika Fukuhara, PhD Student
• Mr. Bakhytzhan Akdavletov, Rotation Student (Jan 2024-)
• Ms. Mary Ludwig, Research Intern (Oct 2023-)
• Mr. Filippo Stocco, Research Intern (Dec 2023-)
• Ms. Mai Yoshioka, Research Unit Administrator (Nov 2023-)
• Dr. Saacnicteh Toledo Patino, Postdoctoral Scholar (-Nov 2023)
• Mr. Stefano Pascarelli, PhD Student (May 2019-April 2023)
• Ms. Sharon Babar, Rotation Student (Sep-Dec 2023)
• Mr. Mauricio andres Rios Maciel, Rotation Student (May-Aug 2023)
• Ms. Trang Vy Bui, Research Intern (Oct 2022- Aug 2023)
• Ms. Diana Nechepurenko, Research Intern (Feb 2023-Aug 2023)
2. Collaborations
2.1 Topic: Beyond the active site: The addition of a remote loop reveals a new complex biological function for chitinase enzymestive site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes
- Type of collaboration: Joint research
- Researchers:
- Professor Adnan Sljoka, Center for Advanced Intelligence Project, RIKEN, Tokyo 103-0027, Japan; Department of Chemistry, York University, Toronto, ON M3J 1P3, Canada
3. Activities and Findings
3.1 Nucleotide-driven specificity of DNA Methyltransfersase
We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l-methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.
3.2 Beyond the active site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes
Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, the evolutionary molecular mechanisms how proteins acquire these loop elements and obtain new function is poorly understood. To address this question, we studied glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity in the catalytic pocket, suggesting an alternative way to design new enzyme function.
4. Publications
4.1 Journals
Gade, M.; Gardner, J.; Jain, P.; Laurino, P.* Nucleotide-driven specifisity of DNA Methyltransfersase ChemBioChem, 2023 24, e2023000
4.2 Books and other one-time publications
4.3 Oral and Poster Presentations
Seminars
1. Laurino P, “insertions and deletions (InDels): Hopeful monsters for protein engineering?” Crick Institute, London UK, 19 April 2023
2. Laurino P, “insertions and deletions (InDels): Hopeful monsters for protein engineering?” Blizard Institute, London UK, 20 April 2023
3. Ochiai Y, “SUPer Rna Ecogii Methyltransferase -SUPREM: an engineered RNA methyltransferases with highly improved stability and activity”. Invited talk hosted by Prof. Joelle N. Pelletier University of Montreal, Montreal, Canada, 11 July 2023
4. Kozone D, “A story of arms races between plants and fungi: Plant chitinase acquires antifungal activity through remote loop evolution.” Hosted by Prof. Gert Bange, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany, 31 July 2023
5. Clifton, B.E., Alcolombri, U., Jackson, C.J., Laurino, P, “Solute-binding proteins: a window to the physiology and evolution of abundant marine bacteria.” Invited lecture presented at Shizuoka University, Shizuoka, Shizuoka Prefecture, Japan, 26 September 2023
6. Laurino P, “Membraneless active droplets mimic features of living systems” Tokyo Tech, Tokyo, Japan, 24 November 2023
7. Ochiai Y, “SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency”. hosted by Dr. Minoru Yoshida, RIKEN, Saitama, Japan, 11 December 2023
Conference Oral Presentations
1. Laurino P, “Membraneless active droplets mimic features of living systems” OIST-Osaka University symposium, Osaka, 29 May 2023
2. Kozone D, “Beyond the active site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes” OIST-Osaka University joint symposium, Osaka, Japan, 29 May 2023
3. Magnus L, “Exploring the FAD binding motif of Rossmann Fold enzymes “Molecular Mechanisms of Evolution, Gordon Research Conference, at Stonhill College in Boston, USA, 26 June 2023
4. Laurino P, “The androgenous enzymes: how nature evolves dual functionalities” Protein evolution meeting, Munster, Germany, 3 July 2023
5. Laurino P, “Indels: The Evolutionary Switches Bridging Protein Functions and Topologies” SPEED symposium, Kyoto, 10 July 2023
6. Ochiai Y, Clifton B.E, Laurino P, “SUPREM: SUPer Rna Ecogii Methyltransferase Engineered by Ancestral Sequence Reconstruction”. The 37th Annual Meeting of Protein Society, MA, USA, 13-16 July 2023
7. Gooch A.S, “Expanding the Substrate Scope of Fluoroacetate Dehalogenase Enzymes via Directed Evolution” SMBE Meeting 2023 Ferrara, Italy, 23-27 July 2003
8. Ochiai Y, Clifton B.E, Laurino P, “Engineering of a non-site-specific RNA methyltransferase by ancestral sequence reconstruction” Hokkaido University mini symposium, Sapporo, Hokkaido Prefecture, Japan, 28 July 2023
9. Laurino P, “Indels: The Evolutionary Switches Bridging Protein Functions and Topologies” Evolutionary biology annual meeting Okinawa, 1 September 2023
10. Muthahari Y. A, “Implementation of Technology and Science in the Modern Era: Career Path in Academia” the International webinar on the Implementation of Technology and Science in the Modern Era, 2 September 2023
11. Ochiai Y, Clifton B.E, Le Coz M, Terenzio M, Laurino P, “SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency”. East-Asia Joint Symposium 2023. Cheonan, South Korea, 24-27 October 2023
12. Kozone D, Oral presentations at Library Design for Protein Engineering 2023, Okinawa, Japan, 6-9 November 2023
13. Ochiai Y, Clifton B.E, Le Coz M, Terenzio M, Laurino P, “SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency”. Library Design and Protein Engineering 2023 (LDPE2023), Okinawa, Japan, 6-9 Nov 2023
14. Clifton, B.E., Alcolombri, U., Jackson, C.J., Laurino, P, “Ultrahigh-affinity transport proteins from ubiquitous marine bacteria: structure, function, and environmental significance” the 61st Annual Meeting of the Biophysical Society of Japan, Nagoya, Aichi Prefecture, Japan, 14 November 2023
15. Laurino P, “insertions and deletions (InDels): Hopeful monsters for protein engineering?” keynote speaker at ASBMB 2023, Canberra, Australia, 15 November 2023
16. Bevilacqua A, “Membranelss active droplets mimic features of living systems” Invited talk at 61st annual meeting of the Biophysical Society of Japan, Nagoya, Japan, November 2023
17. Laurino P, “The androgenous enzymes: how nature evolves dual functionalities” MBSJ, Kobe, Japan, 7 December 2023
18. Bevilacqua A, “Chemotactic interactions drive migration of membrane-less active droplets” hosted by Alessio Scarafoni (University of Milan), Milan, Italy, December 2023
19. Laurino P, “insertions and deletions (InDels): Hopeful monsters for protein engineering?” JST-OIST meeting, Okinawa, 19 January 2024
Conference Poster Presentations
1. Clifton B.E, Uechi G, LaurinoP, “Experimental evolution uncovers the unexpected primary function of a conserved bacterial gene” the ERATO Evolving Symbiosis Project meeting, Okinawa Institute of Science and Technology, Onna, Okinawa Prefecture, Japan, 24 May 2023
2. Gooch A.S, “Expanding the Substrate Scope of Fluoroacetate Dehalogenase Enzymes via Directed Evolution” Joint Poster Session of OIST & ERATO Evolving Symbiosis Project Members (https://groups.oist.jp/ecbsu/event/joint-poster-session-oist-erato-evolving-symbiosis-project-members), OIST, 24 May 2023
3. Ochiai Y, Clifton B.E, Laurino P, “SUPer Rna Ecogii Methyltransferase -SUPREM: an engineered RNA methyltransferases with highly improved stability and activity” 令和５年度日本生化学会九州支部例会 (FY2023 Japanese Biochemical society at Kyushu branch meeting) 24-25 June 2023
4. Ochiai Y, Clifton B.E, Laurino P, “SUPer Rna Ecogii Methyltransferase - SUPREM: an engineered RNA methyltransferases with highly improved stability and activity” RNA Japan Meeting Okinawa, Japan 5-7 July 2023
5. Clifton B.E, Alcolombri, U., Jackson, C.J., Laurino, P, “Functional profiling of transport proteins from ubiquitous marine bacteria reveals mechanisms and global patterns of nutrient uptake” the 23rd Annual Meeting of the Protein Science Society of Japan, Nagoya, Aichi Prefecture, Japan, 7 July 2023
6. Clifton B.E, Alcolombri U, Jackson, C.J., Laurino, P, “Ultra-high affinity transport proteins from ubiquitous marine bacteria reveal mechanisms and global patterns of nutrient uptake” the 37th Annual Symposium of the Protein Society, Boston, MA, 13 July 2023
7. Ochiai Y, Clifton B.E, Laurino P, “SUPREM: SUPer Rna Ecogii Methyltransferase Engineered by Ancestral Sequence Reconstruction”. The 37th Annual Meeting of Protein Society, MA, USA, 13-16 July 2023
8. Kozone D, “Beyond the Active Site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes and the potential remote loop engineering” Society for Molecular Biology and Evolution 2023, Ferrara, Italy, 23-27 July 2023
9. Ochiai Y, Clifton B. E, Laurino P, “SUPREM: SUPer Rna Ecogii Methyltransferase Engineered by Ancestral Sequence Reconstruction” Gordon Research Conferences: Protein Engineering. RI, USA, 23-28 July 2023
10. Bevilacqua A, "Chemotactic interactions drive migration of membraneless active droplets" Annual meeting of Society of Molecular Biology and Evolution, Ferrara, Italy, July 2023
11. Ochiai Y, Clifton B.E, Le Coz M, Terenzio M, Laurino P, “SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency”. East-Asia Joint Symposium 2023. Cheonan, South Korea, 24-27 October 2023
12. Fukuhara E, “"Kinetics of Bacillus subtilis Malate Dehydrogenase and Citrate Synthase in Crowded Protein Droplets" Library Design for Protein Engineering 2023, in OIST, Okinawa, Japan, 6-7 November 2023
13. Jain P, “Engineering Kinase for Enhanced Catalytic Activity with Unconventional Cofactors” Library Design for Protein Engineering 2023, in OIST, Okinawa, Japan, 6-7 November 2023
14. Muthahari Y. A, “Dynamics and Evolution of Uniform Substrate Binders” Library Design for Protein Engineering 2023, in OIST, Okinawa, Japan, 6-7 November 2023
15. Ochiai Y, Clifton B.E, Le Coz M, Terenzio M, Laurino P, “SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency”. Library Design for Protein Engineering 2023, in OIST, Okinawa, Japan, 6-7 November 2023
16. Gooch A.S, “Expanding the Substrate Scope of Fluoroacetate Dehalogenase Enzymes via Directed Evolution” Library Design for Protein Engineering 2023, OIST Mini Symposium, 6-9 November 2023
17. Magnus L,” Exploring the FAD binding motif of Rossmann Fold enzymes” Library Design for Protein Engineering 2023, OIST Mini Symposium, 6-9 November 2023
18. Matthay P, “Exploring the evolutionary landscape of PFAS degradation by Pseudomonas putida” Library Design for Protein Engineering 2023, OIST Mini Symposium, 6-9 November 2023
19. Clifton B.E, Alcolombri U, Jackson, C.J., Laurino, P, “Ultrahigh-affinity transport proteins from ubiquitous marine bacteria: structure, function, and environmental significance” the 61st Annual Meeting of the Biophysical Society of Japan, Nagoya, Aichi Prefecture, Japan, 14 November 2023
20. Fukuhara E, "Unveiling transient complexes: kinetics and structure of the malate dehydrogenase-citrate synthase complex" EMBO Workshop - Computational structural biology, Heidelberg, Germany, 6-9 December 2023
5. Intellectual Property Rights and Other Specific Achievements
Nothing to report
6. Meetings and Events
6.1 Seminar
・ Date: May 22, 2023
・ Venue: OIST campus
・ Speaker: Dr. Yuji Sugita, RIKEN cluster for Pioneering Research
・ Title: Effects of macromolecular crowding on protein dynamics and function
6.2 Seminar
・ Date: Jun 9, 2023
・ Venue: OIST campus
・ Speaker: Prof. Colin Jackson, Australian National University
・ Title: Rugged sequence-activity landscapes in protein evolution"
6.3 Seminar
・ Date: Sep 8-9, 2023
・ Venue: OIST campus
・ Speaker: Prof. Uwe T. Bornsheuer, University of Greifswald
・ Title: Enzyme Discovery & Engineering to Create Suitable Biocatalysts
6.4 Seminar
・ Date: Oct 16, 2023
・ Venue: OIST campus
・ Speaker: Dr. Prof. Makoto Fujita, Distinguished Professor, The University of Tokyo
・ Title: Coordination Self-Assembly: From Origins to the Latest Advances
6.5 LDPE Mini-Symposium “Library Design for Protein Engineering”
・ Date: November 6-9, 2023
・ Venue: OIST campus and Zoom
・ Speakers:
- Dr. Ryoichi Arai, Shinshu University (Japan)
- Dr. Mikael Boden, University of Queensland (Australia)
- Dr. Elizabeth Gillam, University of Queensland (Australia)
- Dr. Sagar Khare, Rutgers University (USA)
- Dr. Dan Thomas Major, Bar-Ilan University (Israel)
- Dr. Joelle Pelletier, University of Montreal (Canada)
- Dr. Jose Sanchez-Ruiz, University of Granada (Spain)
- Dr. Tuck Seng Wong, Sheffield University (UK)
- Dr. Kam Zhang, Riken for Biosystems Dynamics Research (Japan)
6.6 Seminar
・ Date: Dec 4, 2023
・ Venue: OIST campus
・ Speaker: Prof. Hiroaki Suga,The University of Tokyo
・ Title: Pseudo-natural peptides, products and neobiologics for therapeutic applications
6.7 Seminar
・ Date: Jan 12, 2024
・ Venue: OIST campus
・ Speaker: Prof. Fernando Arteaga Arteaga, Hokkaido University
・ Title: Development of photoresponsive self-assembled macrocycle supported on silica nanoparticles for controlled release of Ca2+
6.8 Seminar
・ Date: Jan 15, 2024
・ Venue: OIST campus
・ Speaker: Prof. Emmanuel Levy, the Weizmann Institute of Science
・Title: From sequences to structures, from structures to biology - On homo-oligomers and co-translational assembly
6.9 Seminar
・ Date: Mar 4, 2024
・ Venue: OIST campus
・ Speaker: Dr. Seemay Chou (CEO) and Dr. Prachee Avasthi (CSO)
・ Title: "Evolution as a shortcut to useful inventions"
6.10 Seminar (lead by GS and innovation team)
・ Date: Mar 4, 2024
・ Venue: OIST campus
・ Speaker: Dr. Seemay Chou (CEO) and Dr. Prachee Avasthi (CSO)
・ Title: Arcadia’s journey:building an invention factory
7. Other
Paola was interviewed in Nature Catalysis to discuss the Roz Inclusivity project: "Helping Hands"
