2018 Fiscal Year Research-status Report
Coenzyme engineering for the study of RNA methyltransferase
| Project/Area Number |
18K14656
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
LAURINO Paola 沖縄科学技術大学院大学, タンパク質工学・進化ユニット, 准教授 (90812256)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | coenzyme engineering / RNA methyltransferase |
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| Outline of Annual Research Achievements |
Coenzymes are organic compounds necessary for enzymatic activities. Engineering enzymes to accept synthetic coenzyme can be a key tool for synthetic biology. S-adenosyl methionine (SAM) have been replaced with analogues with minimal structural changes, but replacement of SAM with a synthetic SAM with a different nucleoside is unknown. This modification will allow the engineered enzyme to selectively recognize the synthetic SAM in the presence of natural SAM. In this project, the natural SAM will be replaced with a synthetic SAM and an RNA methyltransferase will be selected by directed evolution to accept the synthetic SAM. The selection system of active RNA methyltransferase variants will be based on in vitro compartmentalization system, which will ensure a successful selection of the active variants.
During this first year we were able to biochemically synthesize few SAM analogs. Analogs were formed by exploiting the promiscuities of human SAM synthetase for nucleotide base, in parallel we also tested 45 designed mutants for a most efficient production but unfortunately known of them turn to be promising for our aim. All of them were tested by the tRNA methyltransferase of our interest (TRM61). Meantime the establishment of the in vitro compartmentalization system was carried on, the system is not trivial but after several attempts we managed to establish it.
In FY19 we will have still to design and generate the library and select for the new coenzyme. Hopefully few good hits will be selected, and we will be able to fully characterize them.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Milestone achieved during this year:
a.Few synthetic SAM analogs have been synthesized biochemically using human SAM synthetase, mainly the nucleoside group has been replaced with other nucleotides. The SAM synthetases reaction has been fully characterized for the reaction with each analog, the Michael mentis parameters has also been retrieved.
b.The tRNA methyltransferase TRM61, has been successfully expressed and purified. The catalytic activity has been tested in vitro, and the enzymatic assay set up.
c.We started to establish the selection system in Vitro. The plasmid construct containing the gene which encode for the tRNA methyltransferase and its substrate has been generated. A control plasmid containing a different gene has also been generated. The emulsion system has been set up and different experiments have been run to check the expression of the methyltransferase by the cell free system. Experiments simulating the selection were successfully carried out using the two plasmids.
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| Strategy for Future Research Activity |
In the following year FY 19 few aims have still to be addressed:
a.Design a library on the catalytic pocket of the tRNA methyltransferase, especially focused on the adenosine binding pocket to allow the accommodation of the new nucleotide.
b.Generate the planned library by PCR assembly.
c.Select by in vitro compartmentalization system for the new coenzyme.
d.Fully characterize the selected variant (Kinetics parameters, x-ray structure, etc)
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| Causes of Carryover |
Not all the budget in FY18 has been used since part of the project has still to be perform in FY19.
The remaining budget will be used to purchase the necessary consumables, solvent, reagents and one UPLC column to accomplish the project in FY19.
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