Abstract
Dimethyl-sulphoniopropionate (DMSP) is produced in abundance by marine phytoplankton, and the catabolism of this compound is an important source of carbon and reduced sulfur for marine bacteria and other organisms. The enzyme DmdD catalyzes the last step in the methanethiol (MeSH) pathway of DMSP catabolism. DmdD is a member of the crotonase superfamily of enzymes, and it catalyzes both the hydration and the hydrolysis of methylthioacryloyl-CoA (MTA-CoA), converting it to acetaldehyde, CO2, MeSH, and CoA. We report here the crystal structure of Ruegeria pomeroyi DmdD free enzyme at 1.5 Å resolution and the structures of the E121A mutant in complex with MTA-CoA and 3-methylmercaptopropionate-CoA (MMPA-CoA) at 1.8 Å resolution. DmdD is a hexamer, composed of a dimer of trimers where the three monomers of each trimer are related by a crystallographic 3-fold axis. The overall structure of this hexamer is similar to those of canonical crotonases. However, the C-terminal loops of DmdD in one of the trimers assume a different conformation and contribute to CoA binding in the active site of a neighboring monomer of the trimer, while these loops in the second trimer are disordered. MTA-CoA is bound deep in the active site in the first trimer, but shows a 1.5 Å shift in its position in the second trimer. MMPA-CoA has a similar binding mode to MTA-CoA in the first trimer. MMPA-CoA cannot be hydrated and is only hydrolyzed slowly by DmdD. Replacement of the sulfur atom in MMPA-CoA with a methylene group abolishes hydrolysis, suggesting that the unique property of the substrate is a major determinant of the hydrolysis activity of DmdD.