S-adenosylhomocysteine Hydrolase Research Articles

Cladribine: not just another purine analogue?

Cladribine was synthesized as a purine analogue drug that inhibited adenosine deaminase. It received FDA approval in the 1980s for treatment of hairy cell leukemia. Given its toxicity towards lymphocytes and its corresponding immunosuppressive effects, it has been studied and found efficacious in a variety of hematologic malignancies and autoimmune conditions, most recently multiple sclerosis. This review highlights pharmacological, toxicological and clinical data for the use of cladribine. It also discusses existing and new mechanisms that may contribute to its unique clinical activity. Emerging data show that in addition to its known purine nucleoside analogue activity, cladribine possesses epigenetic properties, inhibiting S-adenosylhomocysteine hydrolase and DNA methylation. This may contribute to its efficacy and highlights the importance of studying combination therapy with other epigenetic or targeted agents. Clinical trials are underway in a variety of malignant and nonmalignant conditions.

Inherited disorders in the conversion of methionine to homocysteine

During the last decade much important new information relating to the metabolic pathway from methionine to homocysteine has been gained. Interest has been stimulated by the discovery of two novel disorders, glycine N-methyltransferase deficiency and S-adenosylhomocysteine hydrolase deficiency. Another disorder in this pathway, methionine adenosyltransferase deficiency, has been increasingly detected, thanks to the expansion of newborn screening programmes by tandem mass spectrometry technology. These significant steps allow important insight into the pathogenesis of these three disorders, as well as into the mechanisms of damage to various organs (liver, brain, muscle) and point to the relevance of these disorders for crucial biological processes such as methylation, transsulfuration or carcinogenesis in mammals, the pathogenesis of numerous pathological conditions, in particular those associated with hyperhomocysteinaemia, the action and possible toxicity of some drugs or consequences of nutritional variations. This review summarizes current knowledge of three inherited disorders in this metabolic pathway and draws attention to their much broader significance for human health and understanding of important biological processes.

The effects of environmental salinity on trunk kidney proteome of juvenile ayu (Plecoglossus altivelis)

As the life cycle of ayu spans river, brackish and seawater environments, it would be a suitable fish model for studying the responses to salinity changes in aquatic animals. We investigated the effect of salinity on trunk kidney proteome in ayu (Plecoglossus altivelis) using two-dimensional gel electrophoresis and mass spectrometry. The proteins involved in the process of energy metabolism, biosynthesis, DNA methylation and cell differentiation were mainly affected, and 10 significantly changed proteins were identified. Our result showed that isocitrate dehydrogenase (ICD), pyruvate dehydrogenase (E1), O-glycosyl hydrolase, mitochondrial precursor of ATP synthase subunit beta, mitochondrial ferrtin (MtF), retinol binding protein (RBP) were down-regulated, whereas aldehyde dehydrogenase, cytokeratin 1, S-adenosylhomocysteine hydrolase, Cys-Met metabolism PLP-dependent enzyme were up-regulated when ayu transferred from freshwater to brackish water. Partial coding sequences of E1, ICD, MtF and RBP genes were determined, and the effects of salinity on their mRNA expression in ayu trunk kidney were tested by real-time PCR subsequently. Their possible direct or indirect roles in the adaptation of ayu to salinity are discussed.

Mitochondrial S‐Adenosyl‐l‐Methionine Transport is Insensitive to Alcohol‐Mediated Changes in Membrane Dynamics

Alcohol-induced liver injury is associated with decreased S-adenosyl-l-methionine (SAM)/S-adenosyl-l-homocysteine (SAH) ratio and mitochondrial glutathione (mGSH) depletion, which has been shown to sensitize hepatocytes to tumor necrosis factor (TNF). As the effect of alcohol on mitochondrial SAM (mSAM) has been poorly characterized, our aim was to examine the status and transport of mSAM in relation to that of mGSH during alcohol intake. Sprague-Dawley rats were pair fed Lieber-DeCarli diets containing alcohol for 1 to 4 weeks and liver fractionated into cytosol and mitochondria to examine the mSAM transport and its sensitivity to membrane dynamics. We found that cytosol SAM was depleted from the first week of alcohol feeding, with mSAM levels paralleling these changes. Cytosol SAH, however, increased during the first 3 weeks of alcohol intake, whereas its mitochondrial levels remained unchanged. mGSH depletion occurred by 3 to 4 weeks of alcohol intake due to cholesterol-mediated impaired transport from the cytosol. In contrast to this outcome, the transport of SAM into hepatic mitochondria was unaffected by alcohol intake and resistant to cholesterol-mediated perturbations in membrane dynamics; furthermore cytosolic SAH accumulation in primary hepatocytes by SAH hydrolase inhibition reproduced the mSAM depletion by alcohol due to the competition of SAH with SAM for mitochondrial transport. However, alcohol feeding did not potentiate the sensitivity to inhibition by SAH accumulation. Alcohol-induced mSAM depletion precedes that of mGSH and occurs independently of alcohol-mediated perturbations in membrane dynamics, disproving an inherent defect in the mSAM transport by alcohol. These findings suggest that the early mSAM depletion may contribute to the alterations of mitochondrial membrane dynamics and the subsequent mGSH down-regulation induced by alcohol feeding.

A mutant deficient inS-adenosylhomocysteine hydrolase inArabidopsisshows defects in root-hair developmentThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Root-hair development is a process involving the interplay between genetic, hormonal, and environmental factors. An Arabidopsis mutant, referred to as sahh1, was initially recovered from a screen for delayed germination. Molecular characterization of the sahh1 mutant revealed that it contained a T-DNA insertion 82 bp 5′ to the coding sequence of S-adenosylhomocysteine hydrolase 1 (SAHH1, At4g13940), resulting in the reduction of SAHH1 expression. The resulting reduction in expression of SAHH1 produced plants with short, hairless roots, delayed germination, and slow growth. All of these phenotypes were restored to normal by complementing the sahh1 mutant with a full length cDNA. In plants, SAHH1 converts S-adenosylhomocysteine to homocysteine in the activated methyl cycle, and is a precursor for methionine and S-adenosylmethionine. Using the root hairless phenotype of the sahh1 mutant as a visual assay, the effects of SAHH1 deficiency on the synthesis of homocysteine, S-adenosylmethionine, 1-cyclopropane-1-carboxylic acid, and spermidine were investigated.

Ethanol increases histone H3‐K4 methylation and decreases histone H3‐K9 acetylation in conjunction with increased p16 gene expression in the normal colonic epithelial cells

The objective of this study was to determine whether disturbances in one‐carbon metabolism could modify p16 gene expression through histone modifications. NCM460, human colonic epithelial cells, were incubated in media 1) deficient in folate, methionine and choline, 2) with 100μM Adox (adenosine‐2', 3′‐dialdehyde, an S‐adenosylhomocysteine hydrolase inhibitor), and 3) with 100mM ethanol, which inhibits methionine synthesis and reduces methyl group availability. Histone methylation at histone H3 lysine 4 (H3‐K4) and acetylation at lysine 9 (H3‐K9), both known to be increased in active genes, were measured by Western blotting and p16 gene expression by RT‐PCR. Ethanol increased H3‐K4 trimethylation (p=0.005) and decreased H3‐K9 acetylation (p<0.0001) in a time‐dependent fashion. These changes in histone modifications were paralleled with increased p16 expression (p=0.003). Adox decreased H3‐K9 acetylation time‐dependently (p=0.004) without any change in p16 expression. Folate/methionine/choline deficiency didn't produce any changes in this in vitro culture model. This study demonstrates that in this normal colonic cell line, ethanol, which inhibits one‐carbon metabolism, alters H3‐K4 trimethylation and H3‐K9 acetylation in conjunction with an increase in p16 expression.Grant Funding SourceNIH R21 AA016681

TNFα-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafishs-adenosylhomocysteine hydrolase

Hepatic steatosis and liver degeneration are prominent features of the zebrafish ducttrip (dtp) mutant phenotype. Positional cloning identified a causative mutation in the gene encoding S-adenosylhomocysteine hydrolase (Ahcy). Reduced Ahcy activity in dtp mutants led to elevated levels of S-adenosylhomocysteine (SAH) and, to a lesser degree, of its metabolic precursor S-adenosylmethionine (SAM). Elevated SAH in dtp larvae was associated with mitochondrial defects and increased expression of tnfa and pparg, an ortholog of the mammalian lipogenic gene. Antisense knockdown of tnfa rescued hepatic steatosis and liver degeneration in dtp larvae, whereas the overexpression of tnfa and the hepatic phenotype were unchanged in dtp larvae reared under germ-free conditions. These data identify an essential role for tnfa in the mutant phenotype and suggest a direct link between SAH-induced methylation defects and TNF expression in human liver disorders associated with elevated TNFalpha. Although heterozygous dtp larvae had no discernible phenotype, hepatic steatosis was present in heterozygous adult dtp fish and in wild-type adult fish treated with an Ahcy inhibitor. These data argue that AHCY polymorphisms and AHCY inhibitors, which have shown promise in treating autoimmunity and other disorders, may be a risk factor for steatosis, particularly in patients with diabetes, obesity and liver disorders such as hepatitis C infection. Supporting this idea, hepatic injury and steatosis have been noted in patients with recently discovered AHCY mutations.

S-adenosylhomocysteine hydrolase (AHCY) deficiency: Two novel mutations with lethal outcome

This paper reports studies of two novel, allelic missense mutations found in the S-adenosylhomocysteine hydrolase (AHCY) gene from a new case of AHCY deficiency in an infant girl who died at age four months. The mutations lead to replacement of arginine with cysteine (p.Arg49Cys) and aspartic acid with glycine (p.Asp86Gly). Functional analysis of recombinant proteins containing the mutations detected showed that both dramatically reduce AHCY activity. The p.Arg49Cys mutant protein forms intermolecular disulphide bonds, leading to macromolecular structures that can be prevented by reducing agent DTT. The p.Asp86Gly protein tends to form enzymatically inactive aggregates and the loss of a single negative charge as a result of the mutation is involved in enzyme inactivation. We show that replacing Gly86 with negatively charged Glu86 in mutant protein restores enzymatic activity to 70% of wild-type, whereas changing Gly86 to positively charged Lys86 or uncharged Leu86 does not improve enzyme activity, indicating that the negative charge is important for maintenance of such activity. These studies significantly extend knowledge about the importance of residue 86 for AHCY activity. Residue 86 has not been implicated before in this way and the results suggest that the present model of S- adenosylhomocysteine (AdoHcy) hydrolysis may need refinement. Our functional studies provide novel insight into the molecular defect underlying AHCY deficiency and reveal that both low enzyme activity and protein stability of AHCY contribute to the clinical phenotype.

A method for the analysis of domain movements in large biomolecular complexes

A new method for the analysis of domain movements in large, multichain, biomolecular complexes is presented. The method is applicable to any molecule for which two atomic structures are available that represent a conformational change indicating a possible domain movement. The method is blind to atomic bonding and atom type and can, therefore, be applied to biomolecular complexes containing different constituent molecules such as protein, RNA, or DNA. At the heart of the method is the use of blocks located at grid points spanning the whole molecule. The rotation vector for the rotation of atoms from each block between the two conformations is calculated. Treating components of these vectors as coordinates means that each block is associated with a point in a "rotation space" and that blocks with atoms that rotate together, perhaps as part of the same rigid domain, will have colocated points. Thus a domain can be identified from the clustering of points from blocks that span it. Domain pairs are accepted for analysis of their relative movements in terms of screw axes based upon a set of reasonable criteria. Here, we report on the application of the method to biomolecules covering a considerable size range: hemoglobin, liver alcohol dehydrogenase, S-Adenosylhomocysteine hydrolase, aspartate transcarbamylase, and the 70S ribosome. The results provide a depiction of the conformational change within each molecule that is easily understood, giving a perspective that is expected to lead to new insights. Of particular interest is the allosteric mechanism in some of these molecules. Results indicate that common boundaries between subunits and domains are good regions to focus on as movement in one subunit can be transmitted to another subunit through such interfaces.

Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus

An infectious clone (pBR-XJ160) was constructed using the full-length cDNA of the Sindbis-like XJ-160 virus. Two nucleotide mutations, causing amino acid changes at residue 169 from Lys to Arg and at residue 173 from Thr to Ile in the nonstructural protein (nsP) 1 coding region, strongly influenced the infectivity of in vitro-synthesized RNA. We used site-directed mutagenesis to obtain clones encoding a change to Arg at residue 169 of nsP1 (pBR-169), a change to Ile at residue 173 (pBR-173), or both changes (pBR-6973). Infectivity of RNA from pBR-169 was abolished, but viral forms BR-173 and BR-6973 were obtained from pBR-173 and pBR-6973, respectively. Further, BR-173 exhibited higher propagation than BR-XJ160 in cell culture and higher neurovirulence in a suckling mouse model. BR-6973 possessed an intermediate phenotype. BR-173 and BR-6973 showed increased sensitivity to 3-deazaadenosine (3-DZA), which inhibits S-adenosylhomocysteine hydrolase. Thus, mutagenesis at residue 169 in the nsP1 region of XJ-160 is lethal, but mutation at residue 173 from Thr to Ile enhances viral infectivity and neurovirulence and suppresses the lethal effect of the mutation at residue 169. These mutations might be associated with the RNA methyltransferase (MTase) activity of nsP1.

Design and Synthesis of 5''-Iodoneplanocin A and Its Analogues as Potential S-Adenosylhomocysteine Hydrolase Inhibitor

5-Iodoneplanocin A (1) and its analogues 2 and 3 were designed and synthesized as potential SAHH inhibitor via iodocyclopentenol 6, which was prepared using a Michael addition-iodination-elimination process. All final compounds did not show antiviral activity, maybe due to a steric hindrance induced by 5-iodo substituent.

Crystal structures of Mycobacterium tuberculosis S‐adenosyl‐L‐homocysteine hydrolase in ternary complex with substrate and inhibitors

S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 A resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.

Synergistic Pre-Clinical Activity of Combined Epigenetic Therapy with the Novel Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin and Histone Deacetylase Inhibitor Panobinostat against Human AML Cells.

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, regulates the expression of HOX genes and promotes cell proliferation and aggressiveness of neoplastic cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (200 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, inhibits 3Me K27 on H3 while inducing K27 H3 acetylation in the cultured human AML HL60 and OCI-AML3 cells and in primary, patient-derived AML blasts. DZNep treatment also induced the levels of p16, p21, p27 and FBXO32 while depleting cyclin E and HOXA9 levels. Treatment with DZNep induced PARP cleavage activity of the caspases and apoptosis in the cultured and primary AML cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezomib significantly restored EZH2 and SUZ12 levels in the AML cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) also depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e.g., p16 and JunB. Here, we also demonstrate that, as compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2, SUZ12 and EED, more induction of p16, p21 and p27, as well as synergistically induced apoptosis of AML cells (combination indices < 1.0). Additionally, DZNep induced apoptosis of HL-60/LR cells that are resistant to HDACs including PS, as well as sensitized HL-60/LR cells to PS. Taken together, these findings indicate that targeting EZH2 and the PRC2 complex is an effective epigenetic therapy of AML that also overcomes resistance to HDAC inhibitors. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human AML cells, suggesting that this combination may be a promising novel treatment for AML.

Combined Epigenetic Therapy with the Novel Histone Methyl Transferase EZH2 Inhibitor 3-Deazaneplanocin and Histone Deacetylase Inhibitor Panobinostat Exerts Synergistic Activity against Human Mantle Cell Lymphoma Cells

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p16 and JunB. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, as well as promotes cell proliferation and aggressiveness of neoplastic cells. EZH2 is preferentially overexpressed in proliferating but not resting Mantle Cell Lymphoma (MCL) cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (500 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, as well as inhibits 3Me K27 on H3 while inducing K27 H3 acetylation. DZNep treatment also induces the levels of p21, p27, JunB and FBXO32, while depleting cyclin D1 and cyclin E levels in the cultured human MCL Jeko-1, MO2058 and Z138 cells and in primary patient-derived MCL cells. Treatment with DZNep induces PARP cleavage activity of the caspases and apoptosis in the cultured and primary MCL cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezpmib significantly restored EZH2 and SUZ12 levels in the MCL cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e,g, JunB. Here, we also demonstrate that, PS treatment depletes DNMT1 levels and induces JunB levels in cultured MCL cells. As compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2 and SUZ12, but not of DNMT1, more induction of JunB, p21 and p27, as well as synergistically induced apoptosis of cultured MCL cells (combination indices < 1.0). Taken together, these findings indicate that DZNep and PS mediated targeting of EZH2 and the PRC2 complex is an effective epigenetic therapy of MCL, which also results in undermining several molecular determinants of MCL cell proliferation and survival. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human MCL cells, suggesting that this combination may be a promising novel treatment for MCL.

Enzymatic assay for total plasma Cys

Patients with vascular disease and end-stage renal disease have significantly higher concentrations of plasma total Cys (tCys) than do healthy individuals. Described here is a nonradioactive, precise, rapid and sensitive enzymatic colorimetric assay for tCys in plasma samples and is homogeneous in that it avoids separation methods. The tCys assay uses only the recombinant enzymes methionine alpha,gamma-lyase (rMETase) and S-adenosylhomocysteine hydrolase (rSAHH) cloned from Pseudomonas putida and Trichomonas vaginalis, respectively. The rSAHH traps homocysteine and the rMETase thereby produces H(2)S exclusively from Cys. The reaction product, H(2)S, is measured colorimetrically following its reaction with N,N-dibutylphenylenediamine (DBPDA). The procedure takes 3-4 h to complete.

In Silico and Deletion Analysis of Upstream Promoter Fragment of S-Adenosyl Homocysteine Hydrolase (SAHH1) Gene of Arabidopsis Leads to the Identification of a Fragment Capable of Driving Gene Expression in Developing Seeds and Anthers

S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes. A 1585 by fragment upstream to ATG of SAHH1 gene, was fused with a promoter-less β-Glucuronidase (GUS) gene and mobilized into Arabidopsis by Agrobacterium-mediated floral transformation to generate transgenic Arabidopsis. This fragment was found to drive constitutive expression of GUS in T2 progeny of transgenic Arabidopsis. In silico analysis of the promoter region of SAHH1 suggested the presence of several cis-regulatory motifs including seed-specific motifs as well as anther-specific motifs in the 376 by (upstream to TSS of SAHH1) promoter fragment. Based on the partial deletion analysis carried out in the promoter region of SAHH1 (At4gl3940) this 376 by promoter fragment was found to be capable of driving GUS expression in developing seeds and in some anthers/micros pores.

Synthesis of Fluoroneplanocin A

Fluoroneplanocin A, designed as a potent mechanism-based irreversible inhibitor of S-adenosylhomocysteine hydrolase (SAH), is synthesized from D-ribose via a key D-cyclopentenone intermediate. This intermediate is synthesized using a stereoselective Grignard reaction, a ring-closing metathesis (RCM) reaction, and oxidative rearrangement as key steps.

S-adenosylhomocysteine hydrolase downregulation contributes to tumorigenesis

With the idea to discover novel genes involved in proliferation, we have performed a genome-wide loss-of-function genetic screen to identify additional putative tumor suppressor genes. We have previously identified five genes belonging to different biochemical families. In this report, we focused on the study of one of these genes designated S-adenosylhomocysteine hydrolase (SAHH), which has also been previously identified in an independent short hairpin RNA screening. SAHH inactivation confers resistance to both p53 and p16(INK4)-induced proliferation arrest. Interestingly, SAHH inactivation inhibits p53 transcriptional activity and impairs DNA damage-induced transcription of p21(Cip1). Given that SAHH downregulation modulates senescence in primary cells, we also studied SAHH expression in human tumors at the messenger RNA (mRNA) and protein levels. SAHH mRNA was lost in 50% of tumor tissues from 206 patients with different kinds of tumors in comparison with normal tissue counterparts. Moreover, SAHH protein was also affected in some colon cancers. Such findings may be of relevance to cancer research, suggesting that SAHH might be a largely unexplored tumor suppressor.

In Vitro Formation of Homocysteine in Whole Blood in the Presence of Anticoagulants

We read with great interest the letter by Hubner et al. (1) on the stability of plasma sulfur-containing metabolites in the presence of anticoagulants (EDTA and citrate) and a homocysteine stabilizer. These authors observed time-dependent increases in plasma concentrations of homocysteine (Hcy)1 and similar increases in plasma S -adenosylhomocysteine (SAH) in EDTA- and citrate-containing whole blood after 24 h at room temperature. In discussing the mechanisms of these increases, they postulated that plasma Hcy is generated from SAH in erythrocytes by the SAH-hydrolase–catalyzed reaction, and Hcy and SAH leak into plasma from erythrocytes (1). They further noted that the inhibition of SAH hydrolase by citrate results in a smaller Hcy increase compared with EDTA, which they claimed does not inhibit this enzyme. We recently reported the iron-dependent in vitro chemical formation of Hcy from methionine (Met), SAH, and cystathionine, where EDTA or …

Streptococcal Pyrogenic Exotoxin B Cleaves HumanS‐Adenosylhomocysteine Hydrolase and Induces Hypermethioninemia

Group A Streptococcus is a common pathogen that causes pharyngitis, impetigo, myositis, and lethal streptococcal toxic shock syndrome. Streptococcal pyrogenic exotoxin B (SPE B) is strongly associated with the severity of disease. SPE B is a cysteine protease and matures itself by autocatalysis. We found that SPE B was directly associated with human S-adenosylhomocysteine hydrolase (AdoHcyase), an essential factor for a delayed-type immune response. AdoHcyase protein levels and enzymatic activities were significantly higher in human cells infected with the Streptococcus pyogenes SW510 speB mutant strain than in cells infected with the NZ131 wild-type strain. SPE B also inactivated AdoHcyase, shown by a decrease in homocysteine, the main product of AdoHcyase. We found that in vivo and in vitro, SPE B induced hypermethioninemia, which is caused by an AdoHcyase defect. We also found that AdoHcyase is a substrate of SPE B cysteine protease. SPE B, therefore, potentially causes immunosuppression by cleaving AdoHcyase.