Biology subjects

Alcolombri, U.

Publications and source records attributed to Alcolombri, U..

3 recordsLinked to original sources

bioRxiv2018

A redox-regulated type III metacaspase controls cell death in a marine diatom

Programmed cell death (PCD) in marine phytoplankton was suggested as one of the mechanisms that facilitates large scale bloom demise. Yet, the molecular basis for algal PCD machinery is rudimentary. Metacaspases are considered ancestral proteases that regulate cell death, but their activity and role in algae are still elusive. Here we biochemically characterized a recombinant metacaspase 5 from the model diatom Phaeodactylum tricornutum (PtMC5), revealing calcium-dependent protease activity. This activity includes auto-processing and cleavage following arginine. PtMC5 overexpressing cells exhibited higher metacaspase activity and were more sensitive to a diatom-specific infochemical compared to WT cells. Mutagenesis of potential disulfide-forming cysteines decreased PtMC5 activity, suggesting redox regulation. This cysteine pair is widespread in diatom type III metacaspases, but was not found in any other taxa. The characterization of a cell death associated protein in marine phytoplankton will enable deeper understanding of the ecological significance of PCD in bloom dynamics.

microbiology↗

bioRxiv2017

Native or promiscuous? Analyzing putative dimethylsulfoniopropionate lyases using a substrate proofing approach

Enzyme promiscuity is widely spread. Foremost, within superfamilies, the native function of one enzyme is typically observed as promiscuous activity in related enzymes. The native function usually exhibits high catalytic efficiency while promiscuous activities are weak, but this is not always the case. Thus, for certain enzymes it remains questionable whether their currently known activity is native or promiscuous. Dimethylsulfon-iopropionate (DMSP) is an abundant marine metabolite cleaved via {beta}-elimination to release dimethylsulfide (DMS). Eight different gene families have been identified as putative DMSP lyases, 5 of them belonging to the same superfamily (cupin-DLL; see the accompanying paper). Some of these enzymes exhibit very low activity, but this can be due to suboptimal folding or reaction conditions. We developed a substrate profiling approach with the aim of distinguishing native DMSP lyases from enzymes that promiscuously act as DMSP lyases. In a native DMSP lyase, relatively small changes in the structure of DMSP should induce significant activity drops. We thus profiled substrate selectivity by systematically modifying DMSP while retaining reactivity. Three enzymes that exhibit the highest activity with DMSP also exhibited high sensitivity to perturbation of DMSPs structure (Alma, DddY, and DddL). The two enzymes with the weakest DMSP lyase activity also showed the highest crossreactivity (DddQ, DddP). Combined with other indications, it appears that the DMSP lyase activity of DddQ and DddP is promiscuous although their native function remains unknown. Systematic substrate profiling could help identify and assign potential DMSP lyases, and possibly applied to other enzymes.\n\nAbbreviationsDMSP, dimethylsulfoniopropionate; DMS, dimethylsulfide; cupin-DLL, cupin DMSP lyase and lyase-like.\n\nFundingFinancial support by the Estate of Mark Scher, and the Sasson & Marjorie Peress Philanthropic Fund, are gratefully acknowledged. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry.

biochemistry↗

bioRxiv2017

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

Dimethylsulfide (DMS) is released at rates of >107 tons annually and plays a key role in the oceanic sulfur cycle and ecology. Marine bacteria, algae, and possibly other organisms, release DMS via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known belonging to different superfamilies and exhibiting highly variable levels of DMSP lyase activity. DddY shows the highest activity among all reported bacterial lyases yet is poorly characterized. Here, we describe the characterization of recombinant DddY is from different marine bacteria. We found that DddY activity demands a transition metal ion cofactor. DddY also shares two sequence motifs with other bacterial lyases assigned as cupin-like enzymes, DddQ, DddL, DddK, and DddW. These cupin motif residues are essential for DddY activity, as for the other cupin DMSP lyases, and all these enzymes are characterized by a common metal-chelator inhibitor (TPEN). Analysis of all sequences carrying these cupin motifs defined a superfamily: Cupin-DLL (DMSP lyases and lyase-like). The DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily independently along multiple lineages. However, the specific activity levels, genomic context analysis, and systematic profiling of substrate selectivity as described in the accompanying paper, indicate that for only some of these families, most distinctly DddY and DddL, DMSP lyase is the primary, native activity. In other families, foremost DddQ, DMSP lyase seems to be merely a promiscuous activity. The native function of DddQ, and of nearly all members of this newly identified Cupin-DLL superfamily, remains unknown.\n\nAbbreviations\n\nFundingFinancial support by the Estate of Mark Scher, and the Sasson & Marjorie Peress Philanthropic Fund, are gratefully acknowledged. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry.

biochemistry↗