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In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

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In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation International Journal of Molecular Sciences Article In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation Saba Nojoumi 1, Ying Ma 1, Sergej Schwagerus 2,3, Christian P. R. Hackenberger 2,3 and Nediljko Budisa 1,4,* 1 Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Str. 10, D-10623 Berlin, Germany; [email protected] (S.N.); [email protected] (Y.M.) 2 Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, D-12489 Berlin, Germany; [email protected] (S.S.); [email protected] (C.P.R.H.) 3 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Campus Berlin-Buch, Robert-Roessle-Str. 10, D-13125 Berlin, Germany 4 Chair of Chemical Synthetic Biology, Department of Chemistry, University of Manitoba, 144 Dysart Rd, Winnipeg, MB R3T 2N2, Canada * Correspondence: [email protected] or [email protected]; Tel.: +49-30-314-28821 or +1-204-474-9178 Received: 18 April 2019; Accepted: 7 May 2019; Published: 9 May 2019 Abstract: In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings. Keywords: biorthogonal conjugations; deallylation/deprotection; direct sulfhydration/transsulfuration pathway; homocysteine; methionine metabolism; non-canonical amino acids; O-acetyl-homoserine; reprogrammed translation; S-allyl-homocysteine; selective labelling 1. Introduction Canonical amino acid Methionine (Met) is believed to be the most recent addition to the genetic code with the main role of endogenous antioxidants [1] in proteins. Met has very few roles in enzymatic catalytic cycles, whereas in protein folding it behaves similar to the other hydrophobic amino acids [2]. Met contains a unique thioether unit, whose sulphur atom (although often involved in S/π interactions with adjacent aromatic amino acids) can easily be replaced by methylene [3], oxygen [2], selenium [4] and even tellurium [5]. Many non-canonical isosteric analogues and surrogates of Met, which are translationally active, are activated by methionyl-tRNA synthetase (MetRS) with a kinetic turnover similar to those of the native substrate [6]. For this reason, the plasticity of substrate binding in Int. J. Mol. Sci. 2019, 20, 2299; doi:10.3390/ijms20092299 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 2299 2 of 20 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 20 numberwild-type of analogues MetRS can and be surrogates used to co-translate such as metoxini a relativelyne [2], large ethionine number [7], of analogues homopropargylglycine and surrogates [8] andsuch azidohomoalanine as metoxinine [2], [9]. ethionine Recently, [7], no homopropargylglycinen-canonical amino acid [8] (ncAA) and azidohomoalanine S-allyl-L-homocysteine [9]. Recently, (Sahc) wasnon-canonical identified as amino a Met acid analogue (ncAA) S-allyl-that canl-homocysteine be incorporated (Sahc) into was proteins identified in response as a Met analogue to AUG thatsense codonscan be [10]. incorporated In 2013, intoF. Truong proteins [11] in response used a chem to AUGical sense procedure codons [to10 ].synthesise In 2013, F. Sahc Truong and [11 proved] used a its incorporationchemical procedure into a GFP to synthesise variant upon Sahc andfeeding proved Met-auxotrophic its incorporation Escherichia into a GFP coli variant cells. upon In addition feeding to itsMet-auxotrophic translational activity,Escherichia Sahc coli iscells. also In metabolically addition to its active, translational since activity,it serves Sahc as isa alsosubstrate metabolically for Met- adenosyltransferasesactive, since it serves (MATs) as a substrate which forare Met-adenosyltransferasescrucial enzymes in the biosynthesis (MATs) which of the are central crucial enzymesmetabolite in the biosynthesis of the central metabolite S-adenosylmethionine (SAM) [12–15]. Met is also the S-adenosylmethionine (SAM) [12–15]. Met is also the standard starting residue in ribosomal standard starting residue in ribosomal translation, although 60% of these residues are removed by translation, although 60% of these residues are removed by N-terminal processing in E. coli [16]. N-terminal processing in E. coli [16]. It should be emphasised that Sahc is chemically and structurally similar to S-allylcysteine (Sac) It should be emphasised that Sahc is chemically and structurally similar to S-allylcysteine which is naturally abundant in garlic oil [17,18] with a broad range of biological activities. Expectedly, (Sac) which is naturally abundant in garlic oil [17,18] with a broad range of biological activities. natural chemistry of both Sac and Sahc is similar: Sac is known to be a precursor of allicin Expectedly, natural chemistry of both Sac and Sahc is similar: Sac is known to be a precursor of (diallylthiosulfinate)allicin (diallylthiosulfinate) which is which an allelochemic is an allelochemic agent (i.e., agent defence (i.e., defence agent) agent)from garlic from garlic(Allium (Allium sativum L.).sativum Sahc isL.). also Sahc abundant is also in abundant garlic oil in [19] garlic and oil its [19 bioactive] and its bioactiveproperties properties were documented were documented already in 1955already [20]. inTheir 1955 similarity [20]. Their is similarity plausible, is plausible,as Sahc differs as Sahc in di ffchainers in length chain length by only by onlyone onecarbon carbon atom comparedatom compared to Sac, while to Sac, bearing while bearing the same the functional same functional group. group. Both can Both be can synthesised be synthesised in vivoin by vivo simpleby externalsimple addition external addition of allyl ofmercaptan allyl mercaptan to the to thegrow growinging microbial microbial cultures cultures [21]. [21]. However,However, they they are are metabolicallymetabolically different different as as Sac Sac is is a a derivative derivative of Cy Cyss biosynthesis, whereas whereas Sahc Sahc is is a a derivative derivative in in Met Met metabolismmetabolism (Scheme (Scheme 1).1). Finally, Finally, they they are are also also different di fferent in inprotein protein translation. translation. In particular, In particular, Sac Sac is only is recentlyonly recently incorporated incorporated via reprogrammed via reprogrammed translation translation by a bydedicated a dedicated orthogonal orthogonal pair pair for forin-frame in-frame stop codonstop suppression codon suppression [22]. On [22 the]. On other the hand, other Sahc hand, serv Sahces serves as a substrate as a substrate for endogenous for endogenous bacterial bacterial MetRS [11]MetRS and can [11] be and used can for be usedglobal for substi globaltution substitution of Met ofresi Metdues residues in proteins. in proteins. SchemeScheme 1 1.. MetabolismMetabolism and and translational translational activities activities of S-allyl- ofl -cysteineS-allyl- (Sac)L-cysteine and S-allyl- (Sac)l -homocysteineand S-allyl-L- homocysteine(Sahc). Sahc (Sahc). and Sac Sahc differ and in theirSac differ metabolic in their origin metabolic and also origin have diandfferent also modes have different of incorporation modes of incorporationinto recombinant into recombinant proteins. While proteins. Sahc is theWhile replacement Sahc is the for replacement Met residues infor proteins Met residues (recognised in proteins as a (recognisedMet analogue), as a Met Sac isanalogue), not aminoacylated Sac is not by aminoacy natural Cyslated translation by natural machinery Cys translation (i.e., not machinery recognised as(i.e., notCys recognised analogue). as It Cys has recentlyanalogue). been It incorporated has recently into been proteins incorporated [22] by using into anproteins orthogonal [22] pyrrolysylby using an orthogonaltRNA synthetase pyrrolysyl for tRNA in-frame synthetase UAG stop for codon in-frame suppression UAG stop (cAA codon= canonical suppression amino (cAA acid; = ncAAcanonical= aminonon-canonical acid; ncAA amino = non-canonical acid; blue: AUG amino sense acid; codon blue: for AUG Met; purple: sense codon UGU andfor UGCMet; sensepurple: codons UGU for and Cys; red: UAG amber stop codon). UGC sense codons for Cys; red: UAG amber stop codon). The choice of model proteins for incorporation studies with our system is of particular importance, as the presence of Met analogues in protein interiors may be detrimental to their functional integrity [23–26]. For that reason, we have selected a specially designed “hyper stable”
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