Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear Iron-Dependent DMSP Lyase

Abstract

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

DddY is a bacterial dimethylsulfoniopropionate lyase representing a new cupin enzyme superfamily with unknown primary function

“…The mutations H268A, H270A, E274A and H342A all showed complete loss of the activity, as reported for other cupin Ddd+ enzymes such as DddW. 25 The Y277A mutation retained around 20% of the wild-type's activity. However, when Tyr277 was replaced by phenylalanine whose side-chain is similar to tyrosine, enzymatic activity was completely lost (Figure 2C).…”

“…Having collected a range of DddY sequences, we could identify common sequence motifs, including the cupin motifs that are shared by DddQ, DddL, DddK and DddW 13,24,25 . These cupin motifs could also be identified in DddY.…”

Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

“…Anaerobic solutions of BaEFTu-9 were added in aliquots via syringe to the quartz cuvette. The data were fit with Sigma Plot 12.0 (Systat Software Inc., Point Richmond, CA) using eq 1 as described previously 23,30 $$f=f_{\mathrm{o}}+false(f_{\mathrm{m}}-f_{\mathrm{o}}false)\frac{nP+x+K_d-\sqrt[2]{{false(nP+x+K_{d}false)}^2-4\mathrm{italicnPx}}}{2nP}$$where f is the fluorescence signal resulting from binding of the peptide, f o is the signal from a protein solution in the absence of any peptide, f m corresponds to the maximal quenched fluorescence intensity when the peptide is bound, K d is the dissociation constant, P and x are the total protein and ligand concentrations, respectively, and n is the number of binding sites.…”

Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns

“…2 were fit with Sigma Plot 12.0 (Systat Software Inc., Point Richmond, CA) using a ligand-binding model (Equation 1) as described previously (46,47),…”

“…A better approximation of the binding affinity can be obtained by using Equation 1 in cases where the protein concentration is higher than the estimated K d (46,47).…”