Mechanistic Insights into Dimethylsulfoniopropionate Lyase DddY, a New Member of the Cupin Superfamily

Abstract

The marine osmolyte dimethylsulfoniopropionate (DMSP) is one of Earth's most abundant organosulfur molecules. Bacterial DMSP lyases cleave DMSP, producing acrylate and dimethyl sulfide (DMS), a climate-active gas with roles in global sulfur cycling and atmospheric chemistry. DddY is the only known periplasmic DMSP lyase and is present in β-, γ-, δ- and ε-proteobacteria. Unlike other known DMSP lyases, DddY has not been classified into a protein superfamily, and its structure and catalytic mechanism are unknown. Here, we determined the crystal structure of DddY from the γ-proteobacterium Acinetobacter bereziniae originally isolated from human clinical specimens. This structure revealed that DddY contains a cap domain and a catalytic domain with a Zn bound at its active site. We also observed that the DddY catalytic domain adopts a typical β-barrel fold and contains two conserved cupin motifs. Therefore, we concluded that DddY should belong to the cupin superfamily. Using structural and mutational analyses, we identified key residues involved in Zn coordination, DMSP binding and the catalysis of DMSP cleavage, enabling elucidation of the catalytic mechanism, in which the residue Tyr271 of DddY acts as a general base to attack DMSP. Moreover, sequence analysis suggested that this proposed mechanism is common to DddY proteins from β-, γ-, δ- and ε-proteobacteria. The DddY structure and proposed catalytic mechanism provide a better understanding of how DMSP is catabolized to generate the important climate-active gas DMS.

Structure-Function Analysis Indicates that an Active-Site Water Molecule Participates in Dimethylsulfoniopropionate Cleavage by DddK

“…The K m value of DddK determined in this study is lower than that reported previously for the same strain (82 mM) (12), which may be caused by the different buffer systems used. The relatively high K m value, in the millimolar range, is common in many DMSP catabolic enzymes, including DMSP lyases DddP, DddQ, DddY, DddW, and Alma1 and the DMSP demethylase DmdA (11,20,(25)(26)(27)30). The intracellular concentration of DMSP in P. ubique HTCC1062 can reach 180 mM, while the concentration of DMSP in seawater is in the nanomolar level (12), indicating that an efficient transporter for DMSP is required.…”

“…All the other known DMSP lyases generate acrylate and DMS from DMSP (15,18). The algal DMSP lyase Alma1 belongs to the aspartate racemase superfamily (11), DddP is a member of the M24 peptidase family (19), and the five remaining DMSP lyases (DddY, DddK, DddL, DddQ, and DddW) belong to the cupin superfamily and contain a conserved cupin motif (12,15,(20)(21)(22). Cupin superfamily proteins comprise functionally diverse enzymes, which contain a metal ion in their active sites (21,23).…”

“…The advent of biochemical and structural studies on several DMSP lyases has led to the proposal of molecular mechanisms for many of these enzymes (20,(24)(25)(26)(27). Structures of the DMSP lyase DddQ reveal that metal-bound DddQ undergoes a conformational change upon DMSP binding that allows a conserved tyrosine residue to act as a Lewis catalytic base (26).…”

“…Structures of the DMSP lyase DddQ reveal that metal-bound DddQ undergoes a conformational change upon DMSP binding that allows a conserved tyrosine residue to act as a Lewis catalytic base (26). The catalytic mechanisms of DddP and DddY have also been proposed from studies of their crystal structures (20,27,28). Unsurprisingly, being a cupin enzyme, DddY adopts a catalytic mechanism similar to that of DddQ, with a specific tyrosine residue acting as the catalytic base (20).…”

“…The catalytic mechanisms of DddP and DddY have also been proposed from studies of their crystal structures (20,27,28). Unsurprisingly, being a cupin enzyme, DddY adopts a catalytic mechanism similar to that of DddQ, with a specific tyrosine residue acting as the catalytic base (20). In contrast, DddP contains a di-iron core, and an ion-shift catalytic mechanism is proposed (27).…”