Catalytic diversity of cupin domain-containing enzymes

Cupins are a large superfamily of enzymatic and non-enzymatic members that contain a conserved β-barrel domain, or double-stranded β-helix (DSBH) fold. The cupin superfamily is one of the most functionally diverse groups of proteins known to exist. The vast majority of cupins contain a mononuclear metal binding site at the core of the DSBH fold capable of binding different metal ions. One of the largest cupin subfamilies is known as the Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases. Prolyl 4-hydroxylases (P4Hs) belong to the group of Fe(II)/αKG-dependent dioxygenases and catalyze the formation of 4R-hydroxyproline (Hyp) from various proline-containing substrates. The formation of Hyp is an important post-translational modification to many different proteins involved in essential biochemical pathways. Abnormalities in these pathways can lead to diseases such as cancer, fibrosis, respiratory issues, scurvy, and stroke. An Fe(II)/αKG-dependent prolyl hydroxylase from Bacillus anthracis (BaP4H) was investigated to understand its substrate recognition ability and catalytic properties. Novel crystal structures were solved that revealed conformational changes upon substrate binding and key interactions of various ligands in the active site for different catalytic steps. Although the majority of cupin family enzymes catalyze a reaction using iron as an essential cofactor, other metal cofactors can allow the diverse biological transformations carried out by this group of enzymes. A class of enzymes known as dimethylsulfoniopropionate (DMSP) lyases uses different metal ions to catalyze the formation of acrylate and dimethylsulfide (DMS) from DMSP. DMSP is one of the most prevalent and significant molecules to the life and biogeochemistry of the oceans. The products DMS and acrylate are environmentally significant and industrially valuable. DMSP is predominantly catabolized by marine bacteria and can serve different functions. One of the most abundant bacteria in the ocean, Pelagibacter, was determined to contain a DMSP lyase DddK. The DddK catalyzed DMSP lyase activity in the presence of different metal ions has shown that it catalytically prefers Ni(II) compared to other transition metal ions examined. Spectroscopic, site-directed mutagenesis, and crystallographic studies illustrate central residues responsible for metal ion binding and possible roles in transition state stabilization. A greater mechanistic understanding of DMSP lyases will lead to more impactful information about global environmental climate regulation.