A Flexible Platform for the Application of SAM-dependent enzymes

Información del proyecto

AppSAM

Identificador del acuerdo de subvención: 716966

DOI

10.3030/716966

Proyecto cerrado

Fecha de la firma de la CE 15 Marzo 2017

Fecha de inicio 1 Octubre 2017

Fecha de finalización 30 Septiembre 2023

Financiado con arreglo a

• EXCELLENT SCIENCE - European Research Council (ERC)

Coste total

€ 1 499 219,00

Aportación de la UE

€ 1 499 219,00

Coordinado por

ALBERT-LUDWIGS-UNIVERSITAET FREIBURG
Germany

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

Período documentado: 2022-04-01 hasta 2023-09-30

Resumen del contexto y de los objetivos generales del proyecto

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.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

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.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

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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