Marine organisms release dimethylsulfide (DMS) via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known, yet these exhibit highly variable levels of activity. Most assigned bacterial DMSP lyases, including DddK, DddL, DddQ, DddW, and DddY, appear to belong to one, cupin-like superfamily. Here, we attempted to define and map this superfamily dubbed cupin-DLL (DMSP lyases and lyase-like). To this end, we have pursued the characterization of various recombinant DMSP lyases belonging to this superfamily of metalloenzymes, and especially of DddY and DddL that seem to be the most active DMSP lyases in this superfamily. We identified two conserved sequence motifs that characterize this superfamily. These motifs include the metal-ligating residues that are absolutely essential and other residues including an active site tyrosine that seems to play a relatively minor role in DMSP lysis. We also identified a transition metal chelator, N, N, N', N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), that selectively inhibits all known members of the cupin-DLL superfamily that exhibit DMSP lyase activity. A phylogenetic analysis indicated that the known DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily multiple times. However, unusually low specific DMSP lyase activity and genome context analysis suggest that DMSP lyase is not the native function of most cupin-DLL families. Indeed, a systematic profiling of substrate selectivity with a series of DMSP analogues indicated that some members, most distinctly DddY and DddL, are bona fide DMSP lyases, while others, foremost DddQ, may only exhibit promiscuous DMSP lyase activity.

Atmospheric dimethylsulfide (DMS) is massively produced in the oceans by bacteria, algae, and corals. To enable identification of DMS sources, we developed a potent mechanism-based inhibitor of the algal Alma dimethylsulfoniopropionate lyase family that does not inhibit known bacterial lyases. Its application to coral holobiont indicates that DMS originates from Alma lyase(s). This biochemical profiling may complement meta-genomics and transcriptomics to provide better understanding of the marine sulfur cycle.

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Nucleoside-(5'→P) methylenebisphosphonodithioate analogues are bioisosteres of natural nucleotides. The potential therapeutic applications of these analogues are limited by their relative instability. With a view toward improving their chemical and metabolic stability as well as their affinity toward zinc ions, we developed a novel nucleotide scaffold, nucleoside-5'-tetrathiobisphosphonate. We synthesized P1-(uridine/adenosine-5')-methylenebisphosphonodithioate, 2 and 3, and P1,P2-di(uridine/adenosine-5')-methylenebisphosphonodithioate, 4 and 5. Using (1)H and (31)P NMR-monitored Zn(2+)/Mg(2+) titrations, we found that 5 coordinated Zn(2+) by both N7 nitrogen atoms and both dithiophosphonate moieties, whereas 3 coordinated Zn(2+) by an N7 nitrogen atom and Pβ. Both 3 and 5 did not coordinate Mg(2+) ions. (31)P NMR-monitored kinetic studies showed that 3 was more stable at pD 1.5 than 5, with t(1/2) of 44 versus 9 h, respectively, and at pD 11 both showed no degradation for at least 2 weeks. However, 5 was more stable than 3 under an air-oxidizing atmosphere, with t1/2 of at least 3 days versus 14 h, respectively. Analogues 3 and 5 were highly stable to NPP1,3 and NTPDase1,2,3,8 hydrolysis (0-7%). However, they were found to be poor ectonucleotidase inhibitors. Although 3 and 5 did not prove to be effective inhibitors of zinc-containing NPP1/3, which is involved in the pathology of osteoarthritis and diabetes, they may be promising zinc chelators for the treatment of other health disorders involving an excess of zinc ions.

Hybrids of methylenediphosphonotetrathioate and crown-ether (MDPT-CE) were synthesized forming 7-,8-,9-,10- and 13-membered rings. Both 7- and 13-membered ring-containing compounds were found to be highly stable to air-oxidation for at least four weeks. These hybrids bind Zn(II) by both MDPT and CE moieties, forming a 2 : 1 L : Zn(II) complex. Interestingly, the 13-membered ring MDPT-CE showing a high affinity to Zn(II) (Ka 3 ± 0.5 × 10(6) mol(-2) L(2)) does not bind Li(I) or Na(I). The 13-Membered MDPT-CE hybrid is a promising water-soluble, air-stable, high-affinity Zn(II)-chelator, exhibiting selectivity to Zn(II) vs. Mg(II), Na(I), and Li(I).