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Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where “x” is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.

Lysine methylation has been extensively studied in histones, where it has been shown to provide specific epigenetic marks for the regulation of gene expression; however, the molecular mechanism and physiological function of lysine methylation in proteins other than histones remains to be fully addressed. To better understand the substrate diversity of lysine methylation, S-adenosylmethionine (SAM) derivatives with alkyne-moieties have been synthesized. A selenium-based SAM analog, propargylic Se-adenosyl-l-selenomethionine (ProSeAM), has a wide spectrum of reactivity against various lysine methyltransferases (KMTs) with sufficient stability to support enzymatic reactions in vitro. By using ProSeAM as a chemical probe for lysine methylation, we identified substrates for two seven-beta-strand KMTs, METTL21A and METTL10, on a proteomic scale in mammalian cells. METTL21A has been characterized as a heat shock protein (HSP)-70 methyltransferase. Mammalian METTL10 remains functionally uncharacterized, although its ortholog in yeast, See1, has been shown to methylate the translation elongation factor eEF1A. By using ProSeAM-mediated alkylation followed by purification and quantitative MS analysis, we confirmed that METTL21A labels HSP70 family proteins. Furthermore, we demonstrated that METTL10 also methylates the eukaryotic elongation factor EF1A1 in mammalian cells. Subsequent biochemical characterization revealed that METTL10 specifically trimethylates EF1A1 at lysine 318 and that siRNA-mediated knockdown of METTL10 decreases EF1A1 methylation levels in vivo. Thus, our study emphasizes the utility of the synthetic cofactor ProSeAM as a chemical probe for the identification of non-histone substrates of KMTs.

Novel bifunctional catalysts having guanidine and thiourea functional groups were developed for the asymmetric Henry (nitroaldol) reaction. Various structural developments of the catalyst revealed that the compound having an octadecyl-substituted guanidine and thiourea groups linked with a chiral spacer derived from phenylalanine, i.e., 1e, efficiently promoted the Henry reaction. This bifunctional organocatalyst 1e also gave high asymmetric inductions with aliphatic cyclic aldehydes and branched aliphatic aldehydes (82 -90% ee).

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BackgroundG9a and the related enzyme GLP were originally identified as histone lysine methyltransferases and then shown to also methylate several other non-histone proteins.ResultsHere, we performed a comprehensive screen to identify their substrates in mouse embryonic stem cells (mESCs). We identified 59 proteins, including histones and other known substrates. One of the identified substrates, activating transcriptional factor 7-interacting protein 1 (ATF7IP), is tri-methylated at a histone H3 lysine 9 (H3K9)-like mimic by the G9a/GLP complex, although this complex mainly introduces di-methylation on H3K9 and DNA ligase 1 (LIG1) K126 in cells. The catalytic domain of G9a showed a higher affinity for di-methylated lysine on ATF7IP than LIG1, which may create different methylation levels of different substrates in cells. Furthermore, we found that M-phase phosphoprotein 8 (MPP8), known as a H3K9me3-binding protein, recognizes methylated ATF7IP via its chromodomain. MPP8 is also a known component of the human silencing hub complex that mediates silencing of transgenes via SETDB1 recruitment, which is a binding partner of ATF7IP. Although the interaction between ATF7IP and SETDB1 does not depend on ATF7IP methylation, we found that induction of SETDB1/MPP8-mediated reporter-provirus silencing is delayed in mESCs expressing only an un-methylatable mutant of ATF7IP.ConclusionsOur findings provide new insights into the roles of lysine methylation in non-histone substrates which are targeted by the G9a/GLP complex and suggest a potential function of ATF7IP methylation in SETDB1/MPP8-mediated transgene silencing.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0231-z) contains supplementary material, which is available to authorized users.

Chiral metal catalysts have been widely applied to asymmetric transformations. However, the electronic structure of the catalyst and how it contributes to the activation of the substrate is seldom investigated. Here, we report an empirical approach for providing insights into the catalytic activation process in the distorted Ni(II)-catalysed asymmetric [3+2] cycloaddition of α-ketoesters. We quantitatively characterize the bonding nature of the catalyst by means of electron density distribution analysis, showing that the distortion around the Ni(II) centre makes the dz2 orbital partially ‘naked', wherein the labile acetate ligand is coordinated with electrostatic interaction. The electron-deficient dz2 orbital and the acetate act together to deprotonate the α-ketoester, generating the (Λ)-Ni(II)–enolate. The solid and solution state analyses, together with theoretical calculations, strongly link the electronic structure of the centrochiral octahedral Ni(II) complex and its catalytic activity, depicting a cooperative mechanism of enolate binding and outer sphere hydrogen-bonding activation.

Chiral b-amino alcohols are useful building blocks found in various biologically active natural products, pharmaceuticals, chiral auxiliaries, and chiral ligands.[1] Various methods for catalytic enantioselective synthesis of b-amino alcohols have been developed over the past decade, [2] and the catalytic asymmetric nitroaldol (Henry) reaction is an efficient method for providing b-amino alcohols by reduction of the nitro moiety in nitroaldol adducts.[3] Since our first report of the catalytic asymmetric nitroaldol reaction, [4] various chiral catalysts, which are effective with nitromethane as a donor, have been developed.[5] However, diastereo-and enantioselective nitroaldol reactions that use nitroethane and other nitroalkanes as donors are limited. To realize direct nitroaldol reactions, chiral Brønsted base catalysts could deprotonate the a proton of the nitroalkane to generate a metal nitronate, but epimerization of the products must be prevented to achieve high diastereoselectivity under kinetic control. Synselective asymmetric reactions have been established by our group and others; [6] but anti-selective asymmetric reactions required pre-activation of nitroalkanes to silylnitronates [7] to avoid basic conditions. Therefore, a new catalyst for antiselective asymmetric nitroaldol reactions for direct use with nitroalkanes is needed in terms of atom economy.[8] Quite recently, Ooi and co-workers [9] reported an elegant chiral Pspiro triaminoiminophosphorane catalyst for the first direct nitroaldol reaction with excellent anti selectivity, enantioselectivity, and broad substrate generality. [10][11] Considering the importance of anti amino alcohols as precursors for various important pharmaceuticals such as b-adrenoceptor agonists, additional studies of the anti-selective reactions are desirable. Herein, we report a new heterobimetallic Pd/La/1 complex (Scheme 1) for anti-selective nitroaldol reactions, and its application to short syntheses of b-adrenoceptor agonists 2 a·HCl (ritodrine·HCl) and 2 b·HCl.2 a·HCl is a selective b 2 -adrenoceptor agonist, clinically used for the prevention of pre-term birth (Scheme 2), [12] and related compound 2 b·HCl is a selective b 3 -adrenoceptor agonist that provides a new therapeutic for urinary dysfunction.[13] The common chiral anti b-amino alcohol unit (4'-hydroxynorephedrine) in both drugs is key for high biological activity, and we anticipated the anti-nitroaldol reaction to be one of the most straightforward methods for constructing two contiguous stereocenters in the common unit (Scheme 2). 2 a and 2 b could be synthesized by reduction of the nitro group of the anti-nitroaldol adduct with subsequent reductive alkylation of the amine moiety. Initially, we planned to utilize antiselective nitroaldol reactions catalyzed by a Nd/Na/chiral amide complex recently developed by our group, [11] but when used with aldehyde precursors suitable for 2 a and 2 b the reactions resulted in low enantioselectivities of the prod- Scheme 2. Structures and retrosynthesis of (À)-rito...