Evidence for a covalent intermediate in the S-adenosyl-L-methionine-dependent transmethylation reaction catalysed by sirohaem synthase

Abstract

CysG, also known as uroporphyrinogen III methylase and sirohaem synthase (CysG; EC 2.1.1.107), is a multifunctional enzyme that is able to transform uroporphyrinogen III into sirohaem via two S-adenosyl-L-methionine (AdoMet)-dependent transmethylations, an NAD(+)-dependent dehydrogenation and a ferrochelation. The apparent tight binding of AdoMet to this multifunctional enzyme is investigated. The use of a rapid AdoMet binding assay demonstrates that CysG becomes labelled with both [methyl-3H]AdoMet and [carboxyl-14C]AdoMet. Further experiments show that the CysG-AdoMet complex is subsequently able to methylate uroporphyrinogen III. CysG remains associated with the labelled constituents of the AdoMet even after denaturation with urea and SDS/PAGE, suggesting that the AdoMet has become covalently linked to the protein. A rapid examination of some of the other transmethylases involved in corrin biosynthesis reveals that they bind the AdoMet in a similar fashion. A multistep transmethylation mechanism is proposed to explain the observed results.