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.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme‐mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM‐based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM‐based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate‐to‐QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis‐acting QM‐based substrates could be used as activity‐based probes to identify various phospho‐ and phosphono‐ester hydrolases, and potentially other hydrolases.

Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants.