Assigning the Algal Source of Dimethylsulfide Using a Selective Lyase Inhibitor

Alcolombri, Uria; Lei, Lei; Meltzer, Diana; Vardi, Assaf; Tawfik, Dan S.

doi:10.1021/acschembio.6b00844

Cited by 18 publications

(27 citation statements)

References 26 publications

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“…DddY, and DddL cloning, expression, and expression were described in accompanying paper, and Alma1 was expressed as described. 27 , 37 To obtain the kinetic parameters, recombinant DddK, DddW and DddQ enzymes (at 8, 15 and 200 μg/mL respectively were reacted with DMSP at different concentrations up to 10mM for 5 mins (at these enzyme concentrations, rates of release were found to be linear up to 5 min). The amount of, the released DMS was measured to derive the initial rates ( Supplemental Figure S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…The various DMSP analogues were synthesized by Michael addition of the corresponding dialkylsulfide and acrylic acid, or acrylic acid derivative, essentially as described. 37 Typically, the corresponding acrylic acid (1 equivalent) was dissolved in 2M aqueous HCl ( ~ 10 mL for 1 gr), and the respective dialkylsulfide (3-6 molar equivalents) was added portion-wise. The reaction mixture was refluxed at 80°C for 3-12 hours.…”

Section: Methodsmentioning

confidence: 99%

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

Lei

Cherukuri

Meltzer

et al. 2017

Preprint

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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 β-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 DMSP’s 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.AbbreviationsDMSP, dimethylsulfoniopropionate; DMS, dimethylsulfide; cupin-DLL, cupin DMSP lyase and lyase-like.FundingFinancial 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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Lei

Cherukuri

Meltzer

et al. 2017

Preprint

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“…DddY's catalytic efficiency is also considerably higher than that of Alma algal DMSP lyases (0.8 × 10 5 M -1 s -1 for Emiliana huxleyi Alma1 and 2.7 × 10 4 M -1 s -1 for Symbiodinum-A1 Alma1). 6,8 Whilst the 1 st clade that includes AfDddY and DaDddY's clearly encompasses highly active DMSP lyases, the identity of the 2 nd clade of putative DddY's, including the Shewanella, Ferrimonas and Synechococcus genes, is unclear. Upon expression in E. coli, few of the representative genes we tested exhibited weak DMSP lyase activity in crude cell lysates (≤ 14 µM DMS min -1 mg -1 total protein in crude lysate; compared to ~700 for DaDddY and ~400 for AfDddY).…”

Section: Recombinant Dddy's Exhibit Dmsp Lyase Activity With Kcat/km mentioning

confidence: 99%

“…2,4 Also unknown is the relative contribution of different marine species to the global DMS release, or even of bacterial relative to algal mediated release. 6 Several putative bacterial DMSP lyase families were identified and assigned as 'Ddd' (DMSP dependence DMS releasing) genes, 7 as well as one eukaryote family, dubbed Alma, found in organisms such as algae and corals. 8 We aimed at better understanding of the enzymology and phylogenetics of most abundant bacterial DMSP lyases.…”

Section: Introductionmentioning

confidence: 99%

“…However, the vast majority of Cupin-DLLs are obviously not DMSP lyases. However, as exemplified by DddQ, the shared evolutionary origin can give rise to promiscuous DMSP lyase activity.Cupin-DLL enzymes are inhibited by the metal chelator TPEN and unaffected by Br-DMSPWe have recently reported that 2-bromo-3-(dimethylsulfonio)-propionate (Br-DMSP) is a potent and selective mechanism-based inhibitor of the algal Alma DMSP lyases, and does not inhibit any known bacterial Ddd+ enzymes 6. This inhibitor, that covalently modifies the active-site cysteine of the Alma lyases, is a biochemical probe that could detect the presence of Alma DMSP lyase in various marine organisms and environments and assess their contribution to DMS release.…”

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

Lei

Alcolombri

2017

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Dimethylsulfide (DMS) is released at rates of >10 7 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 metalchelator 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.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Aerobic Bacterial Catabolism of Dimethylsulfoniopropionate

Boden

Hutt

2018

Aerobic Utilization of Hydrocarbons, Oils and Lipids

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Dimethylsulfoniopropionate (DMSP) is an organosulfur zwitterion produced by various marine algae and Bacteria as an osmolyte, cryoprotectant, defense molecule, and antioxidant. In the marine environment in particular, it can be degraded by the Bacteria in various ways. In this chapter we cover the biochemistry and physiology of the various pathways of DMSP catabolism, including the three core enzymes DMSP dethiomethylase (EC 4.4.1.3, the so-called DMSP lyase), DMSP demethylase (EC 2.1.1.269), and DMSP CoA transferase/lyase (EC 2.3.1.x). Six isoenzyme classes of DMSP dethiomethylase have been purified and confirmed in marine Bacteria thus far, with a further isoenzyme found in algae that may also occur in Bacteriathese are all discussed in detail. Methodologies for enzyme assays and the synthesis of DMSP hydrochloride are given, including those for radio-and stable-isotope labelling.

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Assigning the Algal Source of Dimethylsulfide Using a Selective Lyase Inhibitor

Cited by 18 publications

References 26 publications

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

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

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

Aerobic Bacterial Catabolism of Dimethylsulfoniopropionate

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