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A Chemo-Enzymatic Approach Towards Gamma-Thio-Nucleoside-5'-Triphosphates

Reference	BB/M024733/1
Principal Investigator / Supervisor	Professor David Hodgson
Co-Investigators / Co-Supervisors	Professor Martin Cann, Dr Martin Schroeder
Institution	Durham University
Department	Chemistry
Funding type	Research
Value (£)	109,470
Status	Completed
Type	Research Grant
Start date	01/02/2016
End date	31/01/2017
Duration	12 months

Abstract

The five technical objectives will be addressed as follows: OBJECTIVE 1: ACCESS ACTIVE NUCLEOSIDE DIPHOPHATE KINASES (NDPKs) His-tagged WT NDPKs and His->Gly active site mutants will be expressed, and activities will be assessed using HPLC, where the rate of equilibration between UTP and ATP will be measured (imidazole will be added to rescue His->Gly mutants). OBJECTIVE 2: EXPLORE IMIDAZOLE THIOPHOSPHORYLATION AND Im RESCUE OF -HIS MUTANT NDPKs Literature shows that His->Gly NDPK competently transfers the gamma-thio-P group of gamma-thio-ATP to imidazole: we envisage the reverse process will proceed smoothly. Thiophosphorylated imidazole will be prepared, exposed to NDPK plus ADP, and gamma-thio-ATP generation will be confirmed by HPLC. Thereafter, imidazole will be phosphorylated in situ and the thiophosphorylation of ADP will be measured. OBJECTIVE 3: EXPLORE DIRECT HIS THIOPHOSPHORYLATION OF WT NDPKs Immobilsed NDPK will be exposed to thiophosphodichloridate ion or thiophosphoryl chloride to generate thiophosphorylated-NDPK. Phosphoryl transfer activity of this NDPK to ADP will be measured by HPLC, and protein thiophosphorylation will be confirmed by MS. These procedures will be repeated concurrently to demonstrate a continuous process. OBJECTIVE 4: EXPLOIT THE MOST PROMISING RESULTS FROM OBJECTIVE 2 & 3 ACROSS A RANGE OF COMMERCIAL NDP SUBSTRATES. HPLC will be used to screen a range of NDP substrates, and products will be compared to reference compounds and/or confirmed using MS and 31-P NMR methods OBJECTIVE 5: CONFIRM UTILITY OF THIO-NTP SYSTEMS NTP-based enzyme systems avilable in each of our labs will be assayed to gauge the 'quality' of representative gamma-thio-NTPs. These will include T7 RNA polymerase (DRWH lab), adenylyl cyclases (MJC lab), and Ire1 systems (MS lab). NDPK will also serve as its own test vehicle, where materials generated by NDPK will be re-assayed in the reverse direction via HPLC.

Summary

Context of Proposed Research Our methods will give a simpler route towards nucleoside-5'-triphosphates (NTPs), which are key molecules in a range of biotechnological and basic research applications. Currently there are two main approaches. The first is readily accessible to biologists, but offers only small amount of material at high cost. The other requires highly trained chemists to provide the materials, where the chemistry is challenging and often unreliable. Our approach represents a new biotechnology Aims and Objectives We aim to provide two related simpler approaches that should allows biologists to access the desired NTPs with relative easy in also allow them to access more useful amounts of these materials in a cost effective manner. Our objectives centre on using an enzyme system, nucleoside diphosphate kinase (NDPK), to facilitate the production of NTPs. Potential Applications and Benefits The specific class of NTP that we will address in this one-year proof-of-concept proposal has a range of applications in determining how enzymes work and also how signaling pathway operate within biological systems. Our approach will benefit research workers in these areas and the specialist chemical companies that prepare NTPs. With our concept proven, we hope to expand our strategy towards a broader range of nucleotide systems, and generate a generic, broad platform towards this class of molecule.

Impact Summary

Academic and industrial researchers will benefit from this research. Our approach will make important research and biotechnology chemicals more readily available to a wider range of workers. We shall protect IP and engage with industrial users in order to allow reap economic benefits. Our research will be published to allow the general research community to benefit from our findings.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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