A Flexible Platform for the Application of SAM-dependent enzymes

Project Information

AppSAM

Grant agreement ID: 716966

DOI

10.3030/716966

Project closed

EC signature date 15 March 2017

Start date 1 October 2017

End date 30 September 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 219,00

EU contribution

€ 1 499 219,00

Coordinated by

ALBERT-LUDWIGS-UNIVERSITAET FREIBURG
Germany

Periodic Reporting for period 4 - AppSAM (A Flexible Platform for the Application of SAM-dependent enzymes)

Reporting period: 2022-04-01 to 2023-09-30

Summary of the context and overall objectives of the project

S-adenosylmethionine (SAM)-dependent enzymes play an important role in all kingdoms of life. SAM is used as a methyl donor for the methylation of small molecules, proteins, and ribonucleic acids by conventional methyltransferases. It is further the cofactor for radical SAM enzymes that can catalyse a multitude of complex reactions, including methylations at unactivated positions. In the wide field of epigenetics, methylations are also an important marker, an example are methylations of nucleobases that are introduced by conventional and radical SAM methyltransferases. The reactions catalysed by SAM-dependent enzymes are highly interesting for technical application, e.g. the synthesis of selectively methylated building blocks for pharmaceuticals. Often, the corresponding synthetically-chemical version of the reaction uses highly toxic and cancerogenic compounds and is rather unselective. In the AppSAM project, we developed strategies to integrate SAM-dependent enzymes in multi-enzyme cascades to facilitate their handling and efficiency for chemical synthesis. These systems are also useful for the mechanistic-functional investigation of SAM-dependent enzymes, e.g. from epigenetic pathways or natural product biosynthesis, using modified (co-)substrates that can be produced in situ. In addition to in vitro multienzyme systems, different in vivo systems have been explored with a range of enzyme/ substrate combinations.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The SAM supply and regeneration strategies developed and optimised in AppSAM are complementary to other described systems and can be used with conventional methyltransferases, radical SAM enzymes and polyamine synthases. Depending on the application, different enzyme preparations, scales, and substrate as well as cosubstrate analogues can be used. Although in general competitive with in upscaled in vitro reactions followed by product isolation, the in vivo systems show different results regarding methylation yield. This seems to depend on the host organism/ enzyme/ substrate combination used, and suggests the necessity to optimise the system for individual applications.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The cyclic in vitro SAM regeneration systems can be used for conventional methyltransferases and other SAM-dependent enzymes (see Figure left: bicyclic regeneration cycle for conventional methyltransferases, right: extended system for multiple types of SAM-dependent enzymes using adenine as central building block). In addition to these in vitro systems, in vivo systems using whole cells are a complementary method. The AppSAM project explored a range of these systems and so contributed to the establishment of a sustainable, flexible platform to use SAM-dependent enzymes for chemical synthesis, which can be easily adapted to new systems. This is currently also explored in industry for selective and sustainable methylation as well as alkylation reactions.

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