S-Adenosyl-L-Homocysteine Hydrolase and Methylation Disorders: Yeast As a Model System

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Biochimica et Biophysica Acta 1832 (2013) 204–215

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Review S-adenosyl-L-homocysteine hydrolase and methylation disorders: Yeast as a model system

Oksana Tehlivets a,⁎, Nermina Malanovic a,1, Myriam Visram a, Tea Pavkov-Keller b, Walter Keller a

a Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010 Graz, Austria b ACIB — Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria

article info abstract

Article history: S-adenosyl-L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological Received 29 June 2012 processes: more than 50 AdoMet-dependent methyltransferases methylate a broad spectrum of cellular com- Received in revised form 14 September 2012 pounds including nucleic acids, proteins and lipids. Common to all AdoMet-dependent methyltransferase re- Accepted 18 September 2012 actions is the release of the strong product inhibitor S-adenosyl-L-homocysteine (AdoHcy), as a by-product Available online 24 September 2012 of the reaction. S-adenosyl-L-homocysteine hydrolase is the only eukaryotic enzyme capable of reversible AdoHcy hydrolysis to adenosine and homocysteine and, thus, relief from AdoHcy inhibition. Impaired Keywords: AdoMet S-adenosyl-L-homocysteine hydrolase activity in humans results in AdoHcy accumulation and severe patho- AdoHcy logical consequences. Hyperhomocysteinemia, which is characterized by elevated levels of homocysteine in Homocysteine blood, also exhibits a similar phenotype of AdoHcy accumulation due to the reversal of the direction of the S-adenosyl-L-homocysteine hydrolase S-adenosyl-L-homocysteine hydrolase reaction. Inhibition of S-adenosyl-L-homocysteine hydrolase is also linked to antiviral effects. In this review the advantages of yeast as an experimental system to understand pathologies associated with AdoHcy accumulation will be discussed. © 2012 Elsevier B.V. Open access under CC BY-NC-ND license.

1. AdoMet — a principal methyl group donor and more strong product inhibitor of AdoMet-dependent methyltransferases and is hydrolyzed by S-adenosyl-L-homocysteine hydrolase to homo- Beyond its role in protein synthesis and structure, methionine, cysteine and adenosine (Fig. 1a). In mammals, homocysteine produced after its activation to AdoMet by methionine adenosyltransferase, by this reaction can be remethylated to methionine by methionine plays a crucial role in many aspects of cellular metabolism. AdoMet is syn- synthase or betaine–homocysteine methyltransferase and, thus, retained thesized in virtually all living organisms [1]. It is the second most widely in the methylation cycle. Alternatively, it can be withdrawn from the used enzyme substrate after ATP [2] and, thus, one of the most versatile methylation cycle by its conversion to cysteine, the precursor of glutathi- biomolecules. Being a high-energy sulfonium compound AdoMet can one, via the two-step transsulfuration pathway. Cystathionine β-synthase serve as a source of all three ligands linked to the sulfur atom. AdoMet is the ﬁrst and rate-limiting enzyme of the latter. Both cystathionine is the methyl group donor used by all organisms in most methyl transfer β-synthase and the enzyme catalyzing the second step of the

reactions [3]. It was also shown to be used in a number of other reactions, transsulfuration pathway, cystathionine γ-lyase, are vitamin B6 re- serving as a source of propylamine for the synthesis of spermidine and quiring enzymes. spermine, amino groups for the synthesis of biotin, or ribosyl groups AdoMet is synthesized by methionine adenosyltransferase from in the synthesis of queuosine, a hypermodiﬁed tRNA, as well as 5′ ATP and methionine. In mammals, methionine can be either ingested deoxyadenosyl radicals required for a number of radical reactions [3,4]. in the diet or formed by methylation of homocysteine using either The fact that under normal conditions more than 90% of total a methyl group of betaine or that of 5-methyltetrahydrofolate. The AdoMet in mammalian cells is used for methylation reactions catalyzed latter is formed by methylenetetrahydrofolate reductase from 5,10- by AdoMet-dependent methyltransferases in which AdoMet donates methylenetetrahydrofolate in a cobalamin-dependent reaction [5]. its methyl group to a large variety of acceptors including nucleic Deﬁciency of methionine synthase that catalyzes remethylation of acids, proteins and lipids underscores the importance of methylation homocysteine to methionine using 5-methyltetrahydrofolate links in downstream reactions of AdoMet [3,5]. AdoHcy, which is released homocysteine and folate metabolism leading to accumulation of as a by-product of AdoMet-dependent methyl transfer reactions, is a methyltetrahydrofolate at the expense of the methylenetetrahydrofolate and tetrahydrofolate pools required for thymidylate and purine biosynthesis [6]. ⁎ Corresponding author. Tel.: +43 316 380 5504; fax: +43 316 380 9854. The enzymes required for AdoMet synthesis, AdoHcy catabolism, E-mail address: [email protected] (O. Tehlivets). 1 Current address: Institute of Biophysics and Nanosystem Research, Austrian Academy homocysteine remethylation and transsulfuration are conserved between of Sciences, Schmiedlstrasse 6, 8042 Graz, Austria. yeast and mammals except for betaine–homocysteine methyltransferase

Fig. 1. AdoMet-dependent methylation: the role of AdoHcy and S-adenosyl-L-homocysteine hydrolase a) in yeast and b) in mammals. Reverse transsulfuration pathway in yeast converts cysteine into homocysteine via cystathionine γ-synthase, Str2, and cystathionine β-lyase, Str3. AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; Hcy, homocysteine; Met, methionine; Sah1, S-adenosyl-L-homocysteine hydrolase in yeast; AHCY, S-adenosyl-L-homocysteine hydrolase in mammals; CTT, cystathionine; Sam1, AdoMet synthetase 1; Sam2, AdoMet synthetase 2; Sam4, AdoMet-homocysteine methyltransferase; Mht1, S-methylmethionine–homocysteine methyltransferase; Met6, methionine synthase; Met25, O-acetylhomoserine sulfhydrylase; Str1, cystathionine γ-lyase; Str2, cystathionine γ-synthase; Str3, cystathionine β-lyase; Str4, cystathionine β-synthase; Gsh1, γ-glutamylcysteine synthetase; MAT, methionine adenosyltransferase; MS, methionine synthase; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine β-synthase; CTH, cystathionine γ-lyase; GCS, γ-glutamylcysteine synthetase. and the reverse transsulfuration pathway (Fig. 1a, b). A further dif- and human AdoMet-dependent methyltransferases grouped by their sub- ference between sulfur metabolism in mammals and in yeast is the strate specificity are compared. The whole list of known and putative presence of the sulfur assimilation pathway in the latter (Fig. 1b); how- yeast and human AdoMet-dependent methyltransferases can be found ever, this pathway can be efficiently eliminated in yeast by the intro- in [18] and [17], respectively. duction of a mutation disrupting the pathway [7].Deficiencies in the The largest group of AdoMet-dependent methyltransferases methyl- enzymes involved in the methylation cycle or homocysteine catabo- ates proteins, predominantly at the ε-amine group of lysine and ω-or lism e.g. cystathionine β-synthase, methionine adenosyltransferase, δ-guanidine groups of arginine as well as on the C-terminal leucine and methylenetetrahydrofolate reductase and S-adenosyl-L-homocysteine isoprenylated cysteine residues. Targets of AdoMet-dependent pro- hydrolase show that many of them are linked to a number of patholo- tein methyltransferases include histones, ribosomal proteins, tran- gies including deregulation of lipid metabolism [7–13], neurological scription and translation factors, signal transduction proteins etc. abnormalities [10,14–16], tumorigenicity [8,9,11], oxidative stress [8] [19–22]. For instance, the SET domain protein lysine methyltransferase and myopathy [14]. family, which makes up 27% of human methyltransferasome and 14% of yeast methyltransferasome, methylates predominantly histones at 2. AdoMet-dependent methyltransferases lysine residues [17,23]. Comparison of a number of histone lysine methyltransferases from various organisms ranging from yeast to Computational analysis of the human proteome revealed 208 human underlines the similarity of their roles in the maintenance of known and putative human AdoMet-dependent methyltransferases normal cellular functions in eukaryotes [24]. that make up about 0.9% of genes in the human genome [17]. Note- Protein arginine methyltransferases methylate in addition to his- worthy, 30% of these proteins have been linked to disease states tones [19,20] numerous non-histone proteins [20,22,25] both in e.g. cancer and mental disorders [17]. Analysis of the yeast proteome re- yeast and in mammals, including heterogeneous nuclear riboproteins vealed 81 known and putative AdoMet-dependent methyltransferases, (hnRNPs) required for eukaryotic transcription elongation, mRNA pro- making up about 1.2% of genes in the yeast genome [18]. The increased cessing and export [26–28]. Further targets for AdoMet-dependent meth- number of AdoMet-dependent methyltransferases in humans as com- ylation are the protein phosphatase 2A (PP2A), which is methylated pared to yeast reflects partial redundancy as well as novel functions pres- at the highly conserved C-terminal leucine of its catalytic C subunits ent in the human methyltransferasome in comparison with yeast [17]. [29], and G proteins, in particular Ras proteins, that are methylated at AdoMet-dependent methyltransferases such as DNA-, glycine-, C-terminal isoprenylcysteine residues [30]. guanidinoacetate- and isoaspartyl methyltransferases are found only in A big group of AdoMet-dependent methyltransferases methylate humans. However, sterol-24C-methyltransferase, Erg6p, is found only in nucleic acids. However, while many AdoMet-dependent methyltrans- yeast. Nevertheless, the majority of AdoMet-dependent methyl- ferases are involved in the methylation of mRNA, rRNA and tRNA transferases are conserved between yeast and humans. In Table 1,yeast both in yeast and in mammals, there are no orthologs to mammalian 206 O. Tehlivets et al. / Biochimica et Biophysica Acta 1832 (2013) 204–215

Table 1 Comparison of yeast and human AdoMet-dependent methyltransferases grouped by substrate speciﬁcity. The whole list of known and putative yeast and human AdoMet-dependent methyltransferases can be found in [18] and [17], respectively.

AdoMet-dependent methyltransferases Biological function/process Yeast Mammalian orthologs

DNA DNA (cytosine-5-)-methyltransferases Gene silencing – DNMT1, DNMT2, DNMT3a, DNMT3b

RNA mRNA (guanine-N7-)-methyltransferases Methylation of the 5′ cap structure of mRNA Abd1 RNMT mRNA (adenosine-N6)-methyltransferases Entry into meiosis Ime4 METTL3 Trimethylguanosine synthases Hypermethylation of m(7)G to the Tgs1 TGS1 m(2,2,7)G 5′ cap of snRNAs, snoRNAs and telomerase TLC1 RNA Probable rRNA (cytosine-5-)-methyltransferases 27S pre-rRNA processing and 60S ribosome Nop2 NOP2 biogenesis rRNA (guanine-N7)-methyltransferases rRNA processing Bud23 WBSCR22 rRNA (adenine-N6,N6-)-dimethyltransferases 18S pre-ribosomal rRNA processing Dim1 DIMT1, TFB1M rRNA (2′-O-ribose)-methyltransferases 27S pre-rRNA and 25S rRNA processing, Spb1 FTSJ1, FTSJ2, FTSJ3 60S ribosomal subunit maturation Mitochondrial rRNA (2′-O-ribose)- Mitochondrial 21S rRNA processing Mrm1, Mrm2 MRM1 methyltransferases tRNA (guanine)-methyltransferases tRNA modification Trm1, Trm5, Trm10, Trm8/Trm82, TRMT1, TRMTL1, TRMT5, RG9MTD2, Trm11/Trm112, Trm12 METTL1, TRMT11, TRMT12 tRNA (uracil-5-)-methyltransferases tRNA modification Trm2, Trm9 TRMT2A, ALKBH8, KIAA145 tRNA (cytosine-5-)-methyltransferases tRNA modification Ncl1 (Trm4) NSUN2, NSUN3, NSUN5 tRNA (adenine-N1-)-methyltransferases Maturation of initiator methionyl-tRNA Trm6/Trm61 TRMT6 tRNA (2′-O-ribose)-methyltransferases tRNA modification Trm3, Trm7, Trm13, Trm44 TARBP1, FTSJ1, CCDC76 tRNA methyltransferases Methylation of N-4 position of yW-86 in Tyw3 TYW3 wybutosine biosynthesis Carboxyl methyltransferases Methylation of the α-carboxy group Ppm2 LCMT2 of yW-72 in wybutosine biosynthesis

Proteins Histone lysine N-methyltransferases Transcriptional elongation and silencing Set1, Set2, Dot1 SET superfamily consisting of SUV39, SET1, SET2, RIZ, SMYD, EZ and SUV4-20 families, SET7/9, SET8, DOT1L Ribosomal protein lysine Ribosome biogenesis Rkm1, Rkm2, Rkm3, Rkm4, Rkm5 SETD6 N-methyltransferases ω-NG-monomethylarginine and Methylation of hnRNPs affecting their activity Hmt1 (Rmt1) PRMT1, PRMT3, PRMT4 (CARM1), PRMT6, asymmetric ω-NG,NG-dimethylarginine and nuclear export; methylation of U1 snRNP PRMT8 methyltransferases protein Snp1p and ribosomal protein Rps2p ω-NG-monomethylarginine and Recruitment and inactivation of Swe1 Hsl7 PRMT5 (JBP1), PRMT7 symmetric ω-NG,NG-dimethylarginine (Wee1 ortholog) during mitotic entry methyltransferases δ-NG-monomethylarginine Methylation of ribosomal protein Rpl12 Rmt2 – methyltransferase Protein histidine methyltransferases 3-Methylhistidine modiﬁcation of ribosomal Hpm1 METTL18 protein Rpl3p N-terminal methyltransferases Methylation of ribosomal proteins Rpl12 and Rps25 Tae1 METTL11A C-terminal leucine carboxyl Methylation of C-terminal leucine of PP2A catalytic Ppm1 LCMT1 methyltransferases subunit, complex formation Protein S-isoprenylcysteine C-terminal methylation of CAAX proteins Ste14 ICMT O-methyltransferases Protein-L-isoaspartate (D-aspartate) Repair of damaged proteins − PCMT1 O-methyltransferase Cytochrome c lysine Trimethylation of cytochrome c, not Ctm1 − N-methyltransferase required for respiratory growth eRF1 methyltransferases Peptidyl-glutamine methylation, Mtq2 N6AMT1 translation release Mrf1methyltransferases Peptidyl-glutamine methylation, Mtq1 HEMK1 mitochondrial translation release

Lipids Phospholipid N-methyltransferases Phosphatidylcholine de novo synthesis Cho2, Opi3 PEMT Sterol 24-C-methyltransferase Ergosterol biosynthesis Erg6 –

Others S-AdoMet-homocysteine Regulation of methionine/AdoMet ratio Sam4 – S-methyltransferase Trans-aconitate methyltransferase Leucine biosynthesis Tmt1 – Uroporphyrin-III C-methyltransferase Siroheme biosynthesis Met1 – Glycine/sarcosine N-methyltransferase Regulation of hepatic AdoMet metabolism – GNMT Guanidinoacetate N-methyltransferase Creatine synthesis – GAMT Ubiquinone methyltransferases Ubiquinone biosynthesis, respiration Coq3, Coq5 COQ3, COQ5 Phenylethanolamine N-methyltransferase Adrenaline (epinephrine) biosynthesis – PNMT Acetylserotonin O-methyltransferase Melatonin biosynthesis – ASMT Histamine N-methyltransferase Histamine degradation – HNMT Catechol O-methyltransferase Degradation of catecholamines – COMT O. Tehlivets et al. / Biochimica et Biophysica Acta 1832 (2013) 204–215 207

Table 1 (continued) AdoMet-dependent methyltransferases Biological function/process Yeast Mammalian orthologs

Indolethylamine N-methyltransferase Tryptamine methylation – INMT Nicotinamide N-methyltransferases Nicotinamide metabolism Nnt1 NNMT

DNA (cytosine-5)-methyltransferases that methylate DNA in cancer, renal insufficiency and diabetic nephropathy [11,14,52–58]. yeast. While methylation of rRNA is crucial for ribosomal pro- The pathological consequences of AdoHcy accumulation as well as the cessing and maturation [31], methylation of tRNA helps to con- molecular mechanisms triggered by elevated AdoHcy levels are largely trol tRNA folding and to ensure decoding specificity and efficiency not understood. Considering the role of AdoMet-dependent meth- [32]. Cap methylation at the 5′-terminal guanosine by mRNA (gua- ylation in biological processes, inhibition of a number of AdoMet- nine-7-)-methyltransferase is an essential process both in yeast and dependent methyltransferase reactions appears to play a central role mammals [33,34]. in AdoHcy-associated pathology. However, evidence of in vivo sensitiv- In addition to proteins and nucleic acids AdoMet-dependent ity of individual AdoMet-dependent methyltransferase reactions as well methyltransferases methylate lipids and a number of small molecules, as the responsiveness of methylation-dependent biological processes to in particular, phosphatidylethanolamine, ubiquinone and nicotinamide AdoHcy accumulation is limited. Since S-adenosyl-L-homocysteine hy- (Table 1). It is noteworthy that de novo biosynthesis of phosphatidylcho- drolase is a crucial physiological regulator of AdoHcy levels we will line from phosphatidylethanolamine catalyzed by AdoMet-dependent discuss dysfunction of S-adenosyl-L-homocysteine hydrolase, its phospholipid methyltransferases is the major AdoMet consumer in evolutional conservation, structural organization and regulation in mice and humans [35,36] as well as in yeast in the absence of choline/ the next sections. ethanolamine supplementation. 4. S-adenosyl-L-homocysteine hydrolase dysfunction 3. The role of S-adenosyl-L-homocysteine hydrolase in AdoMet-dependent methylation Deletion of a locus overlapping the AHCY gene in mouse results in embryonic death [59]. Homozygous insertion mutations in the AdoHcy that is released as a by-product of AdoMet-dependent S-adenosyl-L-homocysteine hydrolase gene in Arabidopsis thaliana methyltransferase reactions, is a potent product inhibitor of most result in zygotic lethality [60]. In contrast, yeast mutants lacking Sah1

AdoMet-dependent methyltransferases with Ki values in the are viable, due to the presence of an alternative pathway for homocys- submicromolar to low micromolar range [37]. AdoHcy was shown to teine synthesis via the sulfur assimilation. Homocysteine that is synthe- inhibit a number of AdoMet-dependent methyltransferases in vitro, sized by this pathway is further used both for the synthesis of cysteine such as mammalian DNA (cytosine-5-)-methyltransferase, mRNA cap and glutathione as well as for the synthesis of methionine and AdoMet (guanine-N7-)-methyltransferase, tRNA (uracil-5-)-methyltransferase, (Fig. 1) [7]. Introduction of an additional mutation in the sulfur as- protein isoprenylcysteine carboxylmethyltransferase and phospholipid similation pathway indeed renders the resulting yeast sah1 mutants methyltransferases [38–45],andin vivo, such as DNA (cytosine-5-)- inviable [7], consistent with an essential function of S-adenosyl-L- methyltransferase and phospholipid methyltransferases [7,46,47]. homocysteine hydrolase. However, not all AdoMet-dependent methyltransferases exhibit S-adenosyl-L-homocysteine hydrolase deficiency in humans, a rare equal sensitivity against AdoHcy. While some AdoMet-dependent genetic disorder, is characterized by up to 150-fold elevated plasma methyltransferases have a Ki value for AdoHcy that is lower than AdoHcy levels with a significant decrease in the AdoMet/AdoHcy ratio the Km value for AdoMet and are especially sensitive to AdoHcy [37], and severe pathological consequences that can lead to death during others, in particular glycine N-methyltransferase, which regulates childhood [14,61–64]. In these patients, three tissues are predominantly AdoMet levels in mammals [1], are only weakly inhibited by AdoHcy affected: muscles, brain and liver, manifested by severe myopathy, slow [48]. Being a potent product inhibitor for most AdoMet-dependent myelination, developmental delay [61–63] as well as mild hepatitis [61] methyltransferases, AdoHcy is a sensitive marker for the cellular and lipid droplet accumulation in the liver [63]. Biochemical analyses methylation status that is reflected by AdoMet/AdoHcy ratio, and can revealed that AHCY deficient patients display 3–20% of mean control be used to predict methylation deficiency e.g. DNA hypomethylation S-adenosyl-L-homocysteine hydrolase activity in the liver, red blood [49]. cells and fibroblasts [61–63]. They exhibit a decrease of phosphatidyl- The only eukaryotic enzyme capable of AdoHcy catabolism both in choline, choline and albumin levels as well as an elevation of creatine yeast and mammals is S-adenosyl-L-homocysteine hydrolase (Sah1 in kinase, alanine transferase, aspartate transaminase [61–63] and lactate yeast, AHCY in mammals), which catalyzes the reversible hydrolysis dehydrogenase [63] levels in the plasma. At the cellular level these of AdoHcy to homocysteine and adenosine. Similarly to AdoHcy ac- patients show a proliferation of smooth and a decrease of rough en- cumulation, interference with S-adenosyl-L-homocysteine hydrolase doplasmic reticulum [61], altered mitochondria [61,63] as well as was shown to affect methylation of different targets of AdoMet- hypermethylation of leukocyte DNA [61–63]. dependent methyltransferases. In particular, inhibition of S-adenosyl- L-homocysteine hydrolase and the resulting increase of AdoHcy levels 5. S-adenosyl-L-homocysteine hydrolase: conservation, structure were shown to be associated with DNA hypomethylation in cultured and catalytic mechanism endothelial cells [46] and to interfere with RNA methylation in cultured lymphocytic leukemia cells [50]. Furthermore, inhibition of S-adenosyl- S-adenosyl-L-homocysteine hydrolase is an exceptionally well- L-homocysteine hydrolase was also shown to result in decreased conserved enzyme exhibiting over 70% identity at the protein level methylation of poly(A)(+) RNA in Xenopus laevis oocytes [51] as well between human and yeast orthologs, and is among the 90 most highly as in inhibition of phospholipid methylation in yeast [7]. In accordance conserved yeast proteins, including actin (90%), ubiquitin (90%), histones with the crucial role of AdoMet-dependent methylation in many (70–90%), ribosomal (70%) and heat shock proteins (70%) [65]. Fig. 2 biological processes, accumulation of AdoHcy and/or S-adenosyl-L- displays a comparison of S-adenosyl-L-homocysteine hydrolase se- homocysteine hydrolase dysfunction is linked to numerous pathologies quences from Archaea, Bacteria and Eucarya. Emphasizing the similarity including neurological and vascular disorders, myopathy, fatty liver, of yeast and human orthologs, many bacterial and some eukaryotic 208 O. Tehlivets et al. / Biochimica et Biophysica Acta 1832 (2013) 204–215

3dhy, PDB 2zj1, PDB 2zj0 and PDB 2ziz [78]), Burkholderia pseudomallei (PDB 3d64), Trypanosoma brucei (PDB 3h9u) and Leishmania major (PDB 3g1u) and the plant Lupinus luteus (PDB 3ond, PDB 3one and PDB 3onf [79]). All structurally characterized Sah1/AHCY proteins except plant S-adenosyl-L-homocysteine hydrolase are tetramers with NADH/ NAD+ cofactor bound in the active site of each subunit [80,81]. Plant S-adenosyl-L-homocysteine hydrolase from L. luteus has been shown to function as a homodimer [79]. The monomeric subunits of the protein contain three domains: N-terminal substrate-binding domain, cofactor-binding domain and C-terminal tail. The C-terminal tails of two subunits reciprocally protrude into opposite subunits and form a part of their cofactor-binding sites [68]. The two dimers then form a tetramer with the four cofactor-binding domains molding the central core of the tetramer structure, and the substrate-binding domains being exposed on the surface. The thermodynamic equilibrium of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase favors the synthesis of AdoHcy from adenosine and homocysteine in vitro [82]. In vivo rapid enzymatic removal of homocysteine (Hcy) and adenosine (Ado) enables the net hydrolysis of AdoHcy [82,83]. The reaction cycle requires Fig. 2. S-adenosyl-L-homocysteine hydrolase: sequence and structural conservation. – + The sequence conservation based on ConSurf calculations [160] using 166 unique reciprocal oxidation reduction of the substrate and NAD [84].In S-adenosyl-L-homocysteine hydrolase sequences is color coded (from blue – identity to the first step, the Ado 3′ hydroxyl group of adenosine (synthetic red – least conservation) and mapped onto the structure of the Pf-SAHH (PDB 1v8b reaction: Ado+Hcy→AdoHcy) or AdoHcy (hydrolytic reaction: [77]). Shown is one monomer of the functional tetrameric S-adenosyl-L-homocysteine AdoHcy→Ado+Hcy) is oxidized to ketone by NAD+ resulting in hydrolase protein in cartoon drawing, the NAD+ cofactor (yellow) is shown in a stick representation and the substrate (Ado) was omitted for clarity. Note that the 40 amino the formation of NADH (Fig. 3). In the next steps the proton is re- acid insertion consisting of 3 helices and a loop (black circle), which is not present in moved from C4′ forming the carbanion intermediate, followed by its mammalian and yeast S-adenosyl-L-homocysteine hydrolases, shows the least degree of cleavage and the release of water or Hcy, respectively. The catalytic conservation. cycle is finished by the addition of Hcy or water to the C4′_C5′ double bond and reduction of the 3′ keto group under regeneration sequences, but not the mammalian and yeast proteins, exhibit an in- of NAD+, forming AdoHcy or Hcy, respectively. Depending on the sertion segment of 40 amino acids of as yet unknown function in the presence of the substrate the protein undergoes large conformational catalytic domain of the enzyme [66]. rearrangements [71,85–87]. Substrate (AdoHcy/Ado) binding induces S-adenosyl-L-homocysteine hydrolase belongs to the large family a structural transition from the open to the closed form of the enzyme, of NAD(P)H/NAD(P)+-binding proteins that share a Rossmann-fold and product (Ado/AdoHcy) release induces the transition back to the [67]. The NAD(P)H/NAD(P)+ binding domain is found in numerous open conformation [85]. dehydrogenases as well as in many other redox enzymes, but is rather unusual for a hydrolase. The atomic structures of S-adenosyl-L- 6. S-adenosyl-L-homocysteine hydrolase: regulation and homocysteine hydrolase from different sources have been resolved: localization Homo sapiens (PDB 1a7a [68], PDB 1li4 [69] and PDB 3nj4 [70]), Rattus norvegicus (PDB 1b3r [71], PDB 1ky4 and PDB 1ky5 [72], PDB 1k0u S-adenosyl-L-homocysteine hydrolase requires NAD+ to maintain [73], PDB 2h5l [74], PDB 1xwf [75] and PDB 1d4f [76]), Plasmodium its quaternary structure and catalytic activity, making the enzyme falciparum (PDB 1v8b [77]), Mycobacterium tuberculosis (PDB 3ce6, PDB sensitive to the redox status of the cell that is reflected by the

Fig. 3. Catalytic activity of S-adenosyl-L-homocysteine hydrolase. O. Tehlivets et al. / Biochimica et Biophysica Acta 1832 (2013) 204–215 209

NAD+/NADH ratio. As described above NAD+ bound to S-adenosyl- levels corresponds to a 10% increase in cardiovascular disease risk L-homocysteine hydrolase is subject to reduction–oxidation cycles [104] and it was shown that about 40% of patients diagnosed with pre- during the reaction [84] (Fig. 3). Moreover, the form of NAD bound mature coronary artery disease, peripheral vascular disease or venous to the enzyme determines binding of adenosine, the substrate for thrombosis exhibit hyperhomocysteinemia [105]. In addition to vascu- the synthetic reaction and the inhibitor of the hydrolytic reaction. It lar diseases hyperhomocysteinemia is also associated with a number of was shown that S-adenosyl-L-homocysteine hydrolase from bovine other pathologies including neurological disorders, cancer, renal insuf- kidney possesses two binding sites for adenosine: a low affinity bind- ficiency, diabetes, birth defects and aging [55,56,104,106–109]. A num- ing site located in the catalytic domain and a high affinity binding site ber of potential mechanisms that are aimed at the explanation of the located in the NAD+/NADH binding domain of the enzyme [88]. The pathological consequences resulting from homocysteine accumulation NAD+ form of S-adenosyl-L-homocysteine hydrolase binds adenosine were proposed [100,105,110]; however, there is still a controversy on with low affinity leading to the formation of 3′-keto-adenosine, re- the underlying metabolic connections [111–113]. duction of the tightly bound NAD+ to NADH and adoption of the Since certain vitamin deficiencies are characterized by mild closed conformation by the enzyme [88]. However, the enzymatically hyperhomocysteinemia the possibility that vitamin supplementation inactive NADH form of the enzyme binds adenosine with high af- could reduce/prevent cardiovascular events was studied in several finity, suggesting a role for adenosine in NADH-induced inhibition large trials. However, it was observed that vitamins, while capable of S-adenosyl-L-homocysteine hydrolase and potential function of of lowering elevated plasma homocysteine levels, do not reduce the the enzyme as intracellular Ado-binding protein [88]. rates of vascular events [114]. This result may be due to the promo- S-adenosyl-L-homocysteine hydrolase was also shown to bind tion of cell proliferation by folic acid through its role in the synthesis copper with high affinity and is indeed the major copper binding pro- of thymidine, the increase of the methylation potential leading to tein in mouse liver [89,90]. It has a similar dissociation constant for changes in gene expression, or an increase in the levels of asymmetric copper as albumin [89] and was suggested to serve as an intracellular dimethylarginine that inhibits the activity of nitric oxide synthase, copper transporter with a putative function in delivering copper to offsetting any positive effect of homocysteine lowering [114]. An ad- superoxide dismutase [91]. Copper inhibits S-adenosyl-L-homocysteine ditional possibility is that a related metabolite rather than homocys- hydrolase activity in a non-competitive manner due to the release of teine could be a trigger of some pathological changes associated NAD+ [92] that results from interruption of subunit interactions, which with elevated homocysteine levels. Since the thermodynamic equilib- leads to a large decrease in the affinity for the NAD+ cofactor [92].How- rium of the S-adenosyl-L-homocysteine hydrolase reaction favors the ever, the significance of regulation of S-adenosyl-L-homocysteine hy- synthesis of AdoHcy [82], the accumulation of hydrolytic products drolase by NAD+, adenosine and copper is not clear. of the reaction, in particular, homocysteine leads to the reversal of In yeast S-adenosyl-L-homocysteine hydrolase is primarily found the physiological direction of the Sah1 catalyzed reaction, resulting in the cytosol [93]. However, AdoMet-dependent methylation takes in AdoHcy synthesis and its accumulation in vivo, as shown both in place in different cellular compartments e.g. nucleus and ER, suggesting mammals as well as in yeast [7,83,115]. Indeed, patients with even that subcellular translocation of the enzyme may play a regulatory role. mild hyperhomocysteinemia (95% CI values for total homocysteine Indeed, S-adenosyl-L-homocysteine hydrolase was found in the nucleus 11.0–14.7 μmol/L) that overlap those of controls (95% CI values 9.8– in association with mRNA (guanine-7-)-methyltransferase and RNA 12.2 μmol/L) exhibit significant elevation of plasma AdoHcy (95% CI polymerase II in transcriptionally active X. laevis oocytes [51,94]. values 32.3–47.7 nmol/L) compared to controls (95% CI values 24.9– S-adenosyl-L-homocysteine hydrolase was also shown to translocate 30.7 nmol/L) [116]. Therefore, even mild hyperhomocysteinemia is into the nucleus in adult mammalian cells under hypoxia conditions characterized by an accumulation of AdoHcy, which may lead to a known to induce transcriptional activity [95]. Localization analyses in decreased methylation potential and the inhibition of a number of A. thaliana also demonstrated that the enzyme is capable of localizing AdoMet-dependent methyltransferases and, thus, contribute to the to the cytoplasm and the nucleus [96], and was proposed to be targeted pathology associated with homocysteine accumulation. to the nucleus in a complex with adenosine kinase, another enzyme re- Notably, the mechanisms that have been proposed to explain the quired for AdoHcy catabolism [96].TheS-adenosyl-L-homocysteine hy- pathological changes associated with elevated homocysteine levels drolase/adenosine kinase complex is, presumably, targeted to the do not address the accumulation of AdoHcy and the inhibition of nucleus by the nuclear localization signal of mRNA (guanine-7-)- AdoMet-dependent methylation in homocysteine-associated pathol- methyltransferase, with which it interacts [96]. ogy, with the exception of an inhibition of DNA methylation [117–119]. However, AdoHcy rather than homocysteine was shown to be a more 7. AdoHcy in hyperhomocysteinemia sensitive indicator of cardiovascular disease [116,120] as well as of renal insufficiency [56] suggesting that AdoHcy is indeed a crucial patholog- The pathological accumulation of homocysteine above the normal ical factor in homocysteine-associated disorders. It was also reported plasma homocysteine level of 10 μmol/L, hyperhomocysteinemia, can that the supplementation with B-vitamins including folate does not be caused by genetic or nutritional factors. Deficiency in cystathionine efficiently lower plasma AdoHcy levels in contrast to homocysteine β-synthase (Fig. 1) or genetic defects of folate and cobalamin metabo- levels in elderly people with hyperhomocysteinemia [121], possibly lisms lead to severe hyperhomocysteinemia characterized by the plasma explaining the failure of homocysteine lowering vitamins to reduce homocysteine levels of more than 100 μmol/L [97] and are rare in com- vascular events mentioned earlier. Furthermore, supporting the patho- parison to mild hyperhomocysteinemia. Mild hyperhomocysteinemia, genic role of AdoHcy, studies in yeast showed that indeed AdoHcy but in turn, may be due to moderate deficiencies of key enzymes involved not homocysteine is more toxic to cells that are deficient in homocys- in homocysteine metabolism, such as the thermolabile variant of teine catabolism [122]. 5,10-methylenetetrahydrofolate reductase gene that has been reported in 5–15% of the general population [98]. Additionally, mild 8. Induced AdoHcy accumulation as a strategy towards antiviral hyperhomocysteinemia may be a result of dietary deficiencies of therapy vitamin cofactors required for homocysteine catabolism — folic acid, vitamins B6 and B12, and is characterized by the plasma total homocys- During the last three decades S-adenosyl-L-homocysteine hydro- teine levels of 15–25 μmol/L [99]. Hyperhomocysteinemia is linked to a lase was intensively studied as a target for antiviral drug design [123]. number of disorders. It is recognized as a strong, independent and Inhibitors that block the enzyme are efficient against many types of causal risk factor for cardiovascular and vascular diseases [100–103]. viruses, including Ebola, that are dependent, in particular, on mRNA In particular, it has been estimated that a 2.5 μM rise in homocysteine methylation [123–125]. In addition to antiviral activity inhibitors 210 O. Tehlivets et al. / Biochimica et Biophysica Acta 1832 (2013) 204–215 targeted at S-adenosyl-L-homocysteine hydrolase show also other ef- take part in the primary metabolism. Phospholipid methylation requires fects of pharmacological importance, for instance, antimalarial and im- three sequential AdoMet-dependent methylation steps to synthesize munosuppressive activity [126,127]. Although AdoHcy accumulation one molecule of phosphatidylcholine (PC) from phosphatidyletha- may be responsible for a number of the effects of inhibitors targeted nolamine (PE) de novo. Whereas in yeast phospholipid methylation at S-adenosyl-L-homocysteine hydrolase, the ability of some of these is the predominant way to synthesize PC (in particular, in the absence inhibitors to undergo metabolic phosphorylation to nucleotides may of choline/ethanolamine in the culture medium), phospholipid meth- account for a part of their biological activities, making it difficult to val- ylation in mammals that occurs primarily in the liver covers only 30% idate the mode of their action [128]. Furthermore, the interference with of hepatic PC synthesis and accounts for estimated 10 μmol and central metabolic pathways associated with high cytotoxicity is an ad- 1.65 mmol PC secreted into bile per day in mice and humans, respec- ditional hindrance in the utilization of S-adenosyl-L-homocysteine hy- tively [36]. Phospholipid methylation is the major AdoMet consumer drolase inhibitors in the potential future clinical applications [125]. in yeast in the absence of choline/ethanolamine supplementation. Nevertheless, suppression of viral replication in plants by controlled Despite of the relatively low contribution of phospholipid de novo syn- downregulation of S-adenosyl-L-homocysteine hydrolase confirms thesis to PC production in mammals this reaction is also the major con- the antiviral activity as a result of AdoHcy accumulation [129]. sumer of AdoMet in mice [35]. Reexamination of the methylation metabolism in humans also revealed that phospholipid methylation, 9. Yeast as a model system to study methylation deficiency but not creatine synthesis as was assumed previously, accounts for the major part of AdoMet being utilized in the human body [36]. Numerous disorders associated with AdoHcy accumulation sug- gest that this metabolite is capable of triggering multiple pathological 10.1. AdoHcy in yeast lipid metabolism mechanisms, presumably via inhibition of different AdoMet-dependent methyltransferases. However, the role of AdoHcy in these diseases as The involvement of S-adenosyl-L-homocysteine hydrolase in the well as the AdoHcy-dependent molecular mechanisms contributing regulation of phospholipid biosynthesis in yeast was found by show- to the consequences inherent to these pathologies is poorly understood. ing that yeast SAH1 is regulated at the transcriptional level in coordina- Of course, understanding the responsiveness of methylation-dependent tion with phospholipid biosynthesis [134]. S-adenosyl-L-homocysteine processes requiring AdoMet-dependent methyltransferases that are hydrolase controls phospholipid de novo methylation in yeast by regu- present only in mammals, e. g. protein L-isoaspartate methyltransferase, lating the hydrolysis of AdoHcy, which is a competitive inhibitor of requires the use of a mammalian model system. However, due to the Cho2 and Opi3 phospholipid methyltransferases that catalyze methyla- complexity of the methylation metabolism its dissection in a multicellular tion of PE to PC [7,45] (Fig. 4). However, not only phospholipid methyl- organism as well as in higher cells is rather difficult. ation, but also a methylation-independent branch of lipid metabolism is The yeast Saccharomyces cerevisiae is a unicellular eukaryote with affected by AdoHcy accumulation in yeast: yeast cells deficient in an about 4 times lower number of genes compared to humans, but AdoHcy catabolism massively accumulate triacylglycerols (TAG) [7]. shares the complexity of the cellular architecture of higher cells. Supporting the causal role of impaired phospholipid methylation in Yeast is especially amenable to experimentation, in particular for ge- the deregulation of TAG metabolism in response to AdoHcy accumula- netic manipulation and whole genome studies. As a result, this model tion, it was found that yeast mutants that are deficient in the enzymatic system is prized with the highest genome annotation level and was activities required for methylation of PE to PC, cho2 and opi3,alsoaccu- successfully used to characterize a number of fundamental biological mulate TAG [7]. Additionally, homocysteine supplementation in yeast processes, including secretion, organelle biogenesis and cell cycle that leads to a 10-fold accumulation of AdoHcy results in the inhibition [130–133]. Yeast exhibits a highly conserved methylation metabolism of phospholipid methylation and accumulation of TAG, confirming a [17,65] and as such is an advantageous system to understand funda- crosstalk between homocysteine and lipid metabolism [7]. mental toxicity of AdoHcy at the cellular level. For instance, yeast In addition to TAG metabolism, transcriptional regulation of phos- mutants capable of reproducible down-regulation of S-adenosyl- pholipid biosynthesis is also affected in mutants impaired in AdoHcy L-homocysteine hydrolase and, thus, in vivo modulation of AdoHcy catabolism. Deficient phospholipid methylation in Sah1 depleted mutant levels can be used as a valuable tool to understand downstream cells unable to hydrolyze AdoHcy, or in cho2 and opi3 mutants, leads to mechanisms triggered by AdoHcy accumulation. Usage of a yeast mu- induced expression of phospholipid biosynthetic (UASINO-responsive) tant that is deficient in homocysteine remethylation to methionine, genes, indicating accumulation of the phospholipid precursor, phos- met6, provides another independent system to understand the role phatidic acid, in the endoplasmic reticulum (ER) [7]. ACC1 encoding of AdoHcy. In this mutant, grown under homocysteine supplementa- acetyl-CoA carboxylase, the first and rate-limiting enzyme of fatty tion, elevated homocysteine leads to AdoHcy synthesis via the rever- acid biosynthesis, is also subject to UASINO-mediated regulation. There- sal of S-adenosyl-L-homocysteine hydrolase reaction resulting in an fore, the inhibition of phospholipid methylation in mutants deficient in accumulation of the latter. However, in this mutant elevated homo- AdoHcy catabolism as well as in cells supplemented with Hcy appear to cysteine cannot be remethylated to methionine, impeding an addi- result in an accumulation of fatty acids and their channeling into TAG, tional increase in AdoMet levels and an elevation of the AdoMet/ which play a crucial role in buffering excess fatty acids [135]. Moreover, AdoHcy ratio. Further advantage of this system is that it uncouples in addition to a deregulated phospholipid and triacylglycerol metabo- the S-adenosyl-L-homocysteine hydrolase function in the regulation lism, down-regulation of SAH1 expression in yeast also impairs sterol of AdoMet-dependent methylation from its potential effect on gluta- synthesis leading to 4-fold elevated squalene levels. This suggests thione levels. Both systems were already shown to help in the identi- an accumulation of early precursors of ergosterol biosynthesis under fication and characterization of critical cellular processes that respond these conditions, presumably as a result of inhibition of sterol to AdoHcy accumulation [7]. In the next section the deregulation of 24-C-methyltransferase (Tehlivets, Kohlwein, unpublished). lipid metabolism, the major consumer of AdoMet both in yeast and in mammals, will be discussed, as a special aspect of AdoHcy toxicity. 10.2. AdoHcy and homocysteine in lipid metabolism in mammals

10. Role of AdoHcy in lipid metabolism AdoHcy also inhibits mammalian phosphatidylethanolamine N-methyltransferase (PEMT) [136,137]. Similarly as in yeast, deﬁciency Changes at the epigenetic level are the most extensively studied of phospholipid methylation in PEMT−/− knockout mice leads to a consequences of methylation deﬁciency [117–119]. However, quanti- rapid decrease of the hepatic PC/PE ratio and accumulation of TAG in tatively the most important AdoMet-dependent methyltransferases the liver, in the absence of choline supplementation [138]. However, O. Tehlivets et al. / Biochimica et Biophysica Acta 1832 (2013) 204–215 211

Fig. 4. Role of AdoHcy and S-adenosyl-L-homocysteine hydrolase in lipid metabolism in yeast. AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; Hcy, homocysteine; Met, methionine; Sah1, S-adenosyl-L-homocysteine hydrolase; CTT, cystathionine; Sam1, AdoMet synthetase 1; Sam2, AdoMet synthetase 2; Sam4, AdoMet-homocysteine methyltransferase; Mht1, S-methylmethionine–homocysteine methyltransferase; Met6, methionine synthase; Met25, O-acetylhomoserine sulfhydrylase; Str1, cystathionine γ-lyase; Str2, cystathionine γ-synthase; Str3, cystathionine β-lyase; Str4, cystathionine β-synthase; Gsh1, γ-glutamylcysteine synthetase; Gro, glycerol; DHAP, dihydroacetone phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol; CDP-DAG, CDP-diacylglycerol; Cho, choline; Etn, ethanolamine; CL, cardiolipin; PG, phosphatidylglycerol; PGP, phosphatidylglycerol phosphate; Glc, glucose; Ins, inositol; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; Cho2, phosphatidylethanolamine methyltransferase catalyzing ﬁrst methylation from PE to PC; Opi3, phospholipid methyltransferase catalyzing two last methylation steps from PE to PC.

TAG accumulation in the livers of these animals is also due to a de- was shown to lead to the activation of the sterol regulatory element- creased TAG secretion from hepatocytes [139]. binding proteins (SREBPs), which function to activate genes encoding Elevated levels of homocysteine are also linked to the deregula- enzymes in cholesterol, fatty acid and triacylglycerol biosynthesis tion of lipid metabolism in mammals. Cystathionine β-synthase−/− and uptake pathways, both in cultured mammalian cell lines as well as knockout mice exhibit severe hyperhomocysteinemia and accumulate in the livers of CBS−/− mice that lack cystathionine β-synthase AdoHcy in all tissues tested [53,140].Thesemutantanimalsshowele- [142,143].Thefinding that overexpression of the ER chaperone GRP78/ vated TAG and nonesterified fatty acid levels in the liver and serum, BiP inhibits homocysteine-induced gene expression [142,144] suggests and develop hepatic steatosis [12]. Another genetic disorder that results that unfolded protein response (UPR) induction plays a direct role in in moderately elevated homocysteine levels, methylenetetrahydrofolate the activation of triacylglycerol and cholesterol biosynthesis in response reductase deficiency, leads to fatty liver development as well as to to homocysteine accumulation in mammals. One of the possible mecha- neuropathology and aortic lipid deposition in mouse model systems nisms responsible for the induction of ER stress and UPR activation in re- [10,141]. Additionally, dietary-induced hyperhomocysteinemia in mice sponse to homocysteine accumulation is the accumulation of saturated also causes fatty liver [142] suggesting that AdoHcy accumulation and fatty acids in membrane phospholipids [145–147]. In accordance, impaired phospholipid methylation are common denominators in overexpression of stearoyl-CoA desaturase was shown to attenuate homocysteine-associated pathologies. palmitate-induced ER stress and protect from lipoapoptosis in mammalian cells [148–150]. 10.3. Activation of UPR and deregulation of unsaturated fatty acid Phospholipid methyltransferases preferentially catalyze synthesis metabolism in AdoHcy-related deficiencies of PC containing di-C16:1 species in yeast both in vitro and in vivo [151]. In line, the PEMT-derived PC pool is also enriched in unsaturated Supporting the role of elevated homocysteine levels in the deregula- fatty acids [152,153]. Supporting the role of PEMT in the metabolism − − tion of lipid metabolism in mammals, homocysteine supplementation of unsaturated fatty acids, PEMT / knockout mice were reported to 212 O. 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