Crystal Structure of DmdD, a Crotonase Superfamily Enzyme That Catalyzes the Hydration and Hydrolysis of Methylthioacryloyl-CoA

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.

Mechanistic insight into 3‐methylmercaptopropionate metabolism and kinetical regulation of demethylation pathway in marine dimethylsulfoniopropionate‐catabolizing bacteria

“…), indicating that these putative genes likely function in ITI_1157 and ISM as they do in R. pomeroyi DMSP demethylation pathway (Reisch et al ., ; ). Since the catalytic mechanisms of DmdA and AcuH have been described (Reisch et al ., ; Schuller et al ., ; Tan et al ., ; Cao et al ., ), the following work is focused on the catalytic mechanisms of DmdB and DmdC to reveal the molecular mechanism for MMPA metabolism in DMSP‐catabolizing Roseobacters.…”

“…A similar phenomenom is proposed in the acrylate catabolic pathway for the AcuI enzyme, which have quite low K m values (1.1–2.8 µM) and very high affinities to the toxic substrate acrylyl‐CoA (Asao and Alber, ; Wang et al ., ). In addition, the very high affinity and catalytic efficiency of DmdD to MTA‐CoA (Tan et al ., ) may imply that MTA‐CoA may be a toxic compound if allowed to accumulate in DMSP‐catabolizing Roseobacters. Indeed, the C α =C β and the carbonyl group of MTA‐CoA (Fig. )…”

Revisiting the methionine salvage pathway and its paralogues

“…In particular, its precursor, dimethylsulphoniopropionate (DMSP), is degraded into methanethiol and also to DMS via promiscuous enzymes of the cupin family that are likely to be relevant in the present context (Lei et al, 2018). DMSP is produced in abundant quantities by marine surfacewater phytoplankton (Tan et al, 2013). The catabolism of this compound is an important source of carbon and reduced sulphur for marine bacteria and other organisms.…”

“…(Malmstrom et al, 2005). The enzyme (methylthio)acryloyl-CoA hydratase, DmdD, a member of the crotonase superfamily, catalyses the last step in the methanethiol pathway of DMSP catabolism: 3-(methylthio)acryloyl-CoA + 2 H 2 O = acetaldehyde + methanethiol + CoA + CO 2 (Tan et al, 2013). This invites us to try and document further this process, as it is likely to have a key role in the general sulphur cycle on Earth.…”

The chemical biology of dimethylsulfoniopropionate

“…A superimposition of the obtained crystallographic data of the DmdA:DMSP and DmdA:FH 4 complexes provided insight into a concerted mechanism of the demethylation reaction with a methyl transfer from DMSP to FH 4 that proceeds with a concomittant proton transfer via water to an unidentified general base. 85 The enzyme crystallises as a hexamer (dimer of trimers) revealing a crystallographic C 3 axis. The mechanism of the first step by DmdA is shown in detail in Scheme 8. such as human phenylethanolamine N-methyltransferase or the protein arginine methyltransferase PRMT3.…”