Arthur M. Geller

Methionine adenosyltransferase: Structure and function

Methionine adenosyltransferase (MAT), a key enzyme in metabolism, catalyzes the synthesis of one of the most important and pivotal biological molecules, S-adenosyl-methionine. In every organism studied thus far, MAT exists in multiple forms; most are encoded by related, but distinct genes. Molecular and immunological studies revealed the presence of considerable conservation in the structure of MAT from different species; however, the various MAT isozymes differ in their physical and kinetic properties in ways that allow them to be regulated differently. Recent studies suggest that human MAT is composed of nonidentical subunits that can assume multiple states of aggregation, each with different kinetic characteristics. The tissue distribution of MAT isozymes and the ability of cells within the same tissue to switch between the different forms of MAT suggest that this mode of regulation is important for cellular function and differentiation. Therefore, understanding the regulation and structure-function relationship of this fascinating enzyme should help us clarify its role in biology and may provide us with tools to effectively manipulate its activity in clinical situations such as cancer, autoimmunity and organ transplantation.

Expression and Functional Interaction of the Catalytic and Regulatory Subunits of Human Methionine Adenosyltransferase in Mammalian Cells

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet). The mammalian MAT II isozyme consists of catalytic α2 and regulatory β subunits. The aim of this study was to investigate the interaction and kinetic behavior of the human MAT II subunit proteins in mammalian cells. COS-1 cells were transiently transfected with pTargeT vector harboring full-length cDNA that encodes for the MAT II α2 or β subunits. Expression of the His-tagged recombinant α2 (rα2) subunit in COS-1 cells markedly increased MAT II activity and resulted in a shift in theK m for l-methionine (l-Met) from 15 μm (endogenous MAT II) to 75 μm(rα2), and with the apparent existence of two kinetic forms of MAT in the transfected COS-1 cell extracts. By contrast, expression of the recombinant β (rβ) subunit had no effect on theK m for l-Met of the endogenous MAT II, while it did cause an increase in both the V maxand the specific activity of endogenous MAT. Co-expression of both rα2 and rβ subunits resulted in a significant increase of MAT specific activity with the appearance of a single kinetic form of MAT (K m = 20 μm). The recombinant MAT II α2 and rβ subunit associated spontaneously either in cell-free system or in COS-1 cells co-expressing both subunits. Analysis of nickel-agarose-purified His-tagged rα2 subunit from COS-1 cell extracts showed that the β subunit co-purified with the α2 subunit. Furthermore, the α2 and β subunits co-migrated in native polyacrylamide gels. Together, the data provide evidence for α2 and β MAT subunit association. In addition, the β subunit regulated MAT II activity by reducing its K m for l-Met and by rendering the enzyme more susceptible to feedback inhibition by AdoMet. We believe that the previously described differential expression of MAT II β subunit may be an important mechanism by which MAT activity can be modulated to provide different levels of AdoMet that may be required at different stages of cell growth and differentiation.

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic α2/α2′ subunits and a noncatalytic β subunit, believed to have a regulatory function. The full-length cDNA that encodes the β subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The β subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native β subunit protein, and to the rβ protein. There is no cross-reactivity between the MAT II α2 or β subunits. None of the anti-β subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no β subunit protein. Interestingly, the rβ subunit associated withE. coli as well as human MAT α subunits. This association changed the kinetic properties of both enzymes and lowered theK m of MAT for l-methionine. Together, the data show that we have cloned and expressed the human MAT II β subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.

Regulation of the human MAT2B gene encoding the regulatory beta subunit of methionine adenosyltransferase, MAT II

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic α2 and a regulatory β subunit. Down-regulation of the MAT II β subunit expression causes a 6–10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the β subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5′-flanking sequences, and elucidated the in vitro and in vivoregulation of its promoter. Transcription of the MAT2B gene initiates at position −203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between −4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position −115 provided full activity; this was attributed to a TATA at −32. The Sp1 site at position +9 was key for the formation of protein·DNA complexes. Mutation of both the Sp1 site at +9 and the TATA at −32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein·DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5′-untranslated sequences play an important role in regulating the MAT2Bgene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels.

CHROMOSOMAL LOCALIZATION AND CATALYTIC PROPERTIES OF THE RECOMBINANT ALPHA SUBUNIT OF HUMAN LYMPHOCYTE METHIONINE ADENOSYLTRANSFERASE

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits α and β. The cDNA sequence of the α subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney α subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3′-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic α subunit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the α subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic α subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the α subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic α/α‘ subunits from HuLy. The K of the recombinant α subunit was 80 μM, which is 20-fold higher than found for the (αα′) β holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the α/α‘ subunits mediate the enzyme catalytic activity and that the β subunit may be a regulatory subunit of extrahepatic MAT.

Differential Regulation of Methionine Adenosyltransferase in Superantigen and Mitogen Stimulated Human T Lymphocytes

Superantigens interact with the T cell receptor for antigen (TCR) and are, therefore, more physiological stimulators of T lymphocytes than nonspecific polyclonal T cell mitogens. The effects of these two classes of T cell stimulators on methionine adenosyltransferase (MAT) andS-adenosylmethionine (AdoMet) levels were investigated. Activation of resting human peripheral blood T lymphocytes by the mitogen phytohemagglutinin (PHA) or the superantigen staphylococcal enterotoxin B (SEB) caused a 3- to 6-fold increase in MAT II specific activity. Although the proliferative response was higher in cultures stimulated with PHA compared with SEB, MAT II activity was comparable in both cultures. Both stimuli caused down-regulation of the MAT 68-kDa λ subunit expression and induced a comparable increase in the expression of the catalytic α2/α2′ subunit mRNA and protein. However, in superantigen-stimulated cells, the expression of the noncatalytic β subunit was down-regulated and virtually disappeared by 72 h post-stimulation; whereas, no change in the expression of this subunit was noted in PHA-stimulated cells. Thus, at 72 h following stimulation, PHA-stimulated cells expressed MAT II α2/α2′ and β subunits while SEB-stimulated cells expressed the α2/α2′ subunits only; the β subunit was no longer expressed in superantigen-stimulated cells. Kinetic analysis of MAT II in extracts of PHA- and SEB-stimulated cells using reciprocal kinetic plots revealed that in the absence of the β subunit theK m of the enzyme for l-methionine (l-Met) was 3-fold higher than in the presence of the β subunit. Furthermore, AdoMet levels were 5-fold higher in cell extracts lacking the β subunit (SEB-stimulated cell extracts) compared with extracts containing MAT II α2/α2′ and β subunits. We propose that the increased levels of AdoMet in superantigen-stimulated cells may be attributed to the absence of the β subunit, which seems to have rendered MAT II less sensitive to product feedback inhibition by (−)AdoMet. The data suggest that the β subunit of MAT II, which has no catalytic activity, may be a regulatory subunit that imparts a lowerK m for l-Met but increases the sensitivity to feedback inhibition by AdoMet. The down-regulation of the β subunit, which occurred when T cells were stimulated via the TCR, may be an important mechanism to regulate AdoMet levels at different stages of T cell differentiation under physiological conditions.

Purification and properties of rat lens methionine adenosyltransferase

Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIβ subunit in Jurkat cells and accordingly shifted the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{m{\ }L-\mathrm{Met}}\) \end{document} of the enzyme 10–15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 μm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{m{\ }L-\mathrm{Met}}\) \end{document} due to MAT-IIβ ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rγnull humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIβ failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIβ ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Creation of a functional S-nitrosylation site in vitro by single point mutation.

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild‐type and mutant methionine adenosyltransferase were incubated with S‐nitrosoglutathione the activity of the wild‐type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S‐nitrosylated upon incubation with the nitric oxide donor. Treatment of the S‐nitrosylated mutant enzyme with glutathione removed most of the S‐nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S‐nitrosylation sites can develop from existing structures without drastic or large‐scale amino acid replacements.