Biochemical studies of the radical SAM enzyme HydG

Abstract

Radical SAM (RSAM) enzymes are a large family of enzymes which catalyse the reductive cleavage of S-adenosylmethionine, bound at the unique iron of a [4Fe-4S] cluster, to generate a highly reactive deoxyadenosyl radical. This highly reactive intermediate abstracts a hydrogen atom from a substrate to initiate a diverse range of radical mechanisms. RSAM enzymes are responsible for the formation of many organic cofactors, nucleic acid modification, such as methylation of aromatic carbon centres, formation of antibiotics, DNA dimer repair/isomerisation and metallocofactor assembly in the maturation of [FeFe]-hydrogenase enzymes. RSAM enzymes share structural similarities, such as a C-terminal [4Fe-4S] cluster, coordinated by three protein derived cysteine residues (denoted by the CxxxCxxC sequence motif) and at the active site a triose-phosphate isomerase barrel fold. Despite the many thousands of RSAM enzyme sequences, comparatively few have been characterised and the majority possess unknown structures and functions. This project aims to further characterise the RSAM enzyme HydG, one of three maturase enzymes involved in the assembly of the [FeFe] hydrogenase H-cluster. HydG is the most investigated of the maturase enzymes with its substrates and structure well resolved, the product of HydG, proposed to be a [FeII(CO)2(CN)Cys]- metallosynthon has been observed spectroscopically bound to the enzyme auxiliary cluster, and synthesised to become an active substrate of HydE but not been characterised upon release from HydG. In this work Thermoanaerobacter italicus derived HydG has been heterologously expressed, purified, and chemically reconstituted to produce active enzyme. HydG activity assays have been optimised and co-crystallisation screening experiments explored.

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ORCID for Matthias Baud: orcid.org/0000-0003-3714-4350

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