Isolation and Assays of Bacterial Dimethylsulfoniopropionate Lyases.

The organosulfur metabolite dimethylsulfoniopropionate (DMSP) and its enzymatic breakdown product dimethyl sulfide (DMS) have important implications in the global sulfur cycle and in marine microbial food webs. Enormous amounts of DMSP are produced in marine environments where microbial communities import and catabolize it via either the demethylation or the cleavage pathways. The enzymes that cleave DMSP are termed "DMSP lyases" and generate acrylate or hydroxypropionate, and ~107tons of DMS annually. An important environmental factor affecting DMS generation by the DMSP lyases is the availability of metal ions as these enzymes use various cofactors for catalysis. This chapter summarizes advances on bacterial DMSP catabolism, with an emphasis on various biochemical methods employed for the isolation and characterization of bacterial DMSP lyases. Strategies are presented for the purification of DMSP lyases expressed in bacterial cells. Specific conditions for the efficient isolation of apoproteins in Escherichia coli are detailed. DMSP cleavage is effectively inferred, utilizing the described HPLC-based acrylate detection assay. Finally, substrate and metal binding interactions are examined using fluorescence and UV-visible assays. Together, these methods are rapid and well suited for the biochemical and structural characterization of DMSP lyases and in the assessment of uncharacterized DMSP catabolic enzymes, and new metalloenzymes in general.

New Mechanistic Insight from Substrate- and Product-Bound Structures of the Metal-Dependent Dimethylsulfoniopropionate Lyase DddQ.

The marine microbial catabolism of dimethylsulfoniopropionate (DMSP) by the lyase pathway liberates ∼300 million tons of dimethyl sulfide (DMS) per year, which plays a major role in the biogeochemical cycling of sulfur. Recent biochemical and structural studies of some DMSP lyases, including DddQ, reveal the importance of divalent transition metal ions in assisting DMSP cleavage. While DddQ is believed to be zinc-dependent primarily on the basis of structural studies, excess zinc inhibits the enzyme. We examine the importance of iron in regulating the DMSP ß-elimination reaction catalyzed by DddQ as our as-isolated purple-colored enzyme possesses ∼0.5 Fe/subunit. The UV-visible spectrum exhibited a feature at 550 nm, consistent with a tyrosinate-Fe(III) ligand-to-metal charge transfer transition. Incubation of as-isolated DddQ with added iron increases the intensity of the 550 nm peak, whereas addition of dithionite causes a bleaching as Fe(III) is reduced. Both the Fe(III) oxidized and Fe(II) reduced species are active, with similar kcat values and 2-fold differences in their Km values for DMSP. The slow turnover of Fe(III)-bound DddQ allowed us to capture a substrate-bound form of the enzyme. Our DMSP-Fe(III)-DddQ structure reveals conformational changes associated with substrate binding and shows that DMSP is positioned optimally to bind iron and is in the proximity of Tyr 120 that acts as a Lewis base to initiate catalysis. The structures of Tris-, DMSP-, and acrylate-bound forms of Fe(III)-DddQ reported here illustrate various states of the enzyme along the reaction pathway. These results provide new insights into DMSP lyase catalysis and have broader significance for understanding the mechanism of oceanic DMS production.

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

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.