Molecular genetics of hepatic methionine adenosyltransferase deficiency

Pharmacology & Therapeutics 85 (2000) 1–9

Associate editor: P.K. Chiang

Molecular genetics of hepatic methionine adenosyltransferase deficiency Janice Yang Chou* Heritable Disorders Branch, National Institute of Child Health and Human Development, Building 10, Room 9S241, National Institutes of Health, Bethesda, MD 20892-1830, USA

Abstract Hepatic methionine adenosyltransferase (MAT) deficiency is caused by mutations in the human MAT1A gene that abolish or reduce hepatic MAT activity that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. This genetic disorder is characterized by isolated persistent hypermethioninemia in the absence of cystathionine b-synthase deficiency, tyrosinemia, or liver disease. Depending on the nature of the genetic defect, hepatic MAT deficiency can be transmitted either as an autosomal recessive or dominant trait. Genetic analyses have revealed that mutations identified in the MAT1A gene only partially inactivate enzymatic activity, which is consistent with the fact that most hepatic MAT-deficient individuals are clinically well. Two hypermethioninemic individuals with null MAT1A mutations have developed neurological problems, including brain demyelination, although this correlation is by no means absolute. Presently, it is recommended that a DNA-based diagnosis should be performed for isolated hypermethioninemic individuals with unusually high plasma methionine levels to assess if therapy aimed at the prevention of neurological manifestations is warranted. Published 1999 by Elsevier Science Inc. All rights reserved. Keywords: Methionine adenosyltransferase I/III deficiency; S-adenosylmethionine; Genetic mutations; Autosomal recessive or dominant inheritance Abbreviations: AdoMet, S-adenosylmethionine; hMAT, human methionine adenosyltransferase; MAT, methionine adenosyltransferase.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Methionine adenosyltransferase and S-adenosylmethionine . . . . . . . . . . . . . . . . . . . . . . . . 3. The human methionine adenosyltransferase genes and the encoded isozymes . . . . . . . . . . 4. The molecular basis of hepatic methionine adenosyltransferase deficiency . . . . . . . . . . . . 4.1. The hypermethioninemic phenotype can be transmitted both as autosomal recessive and dominant traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The phenotype of a null hMAT1A mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Newborn children are routinely screened for hypermethioninemia as an indicator of hyperhomocyst(e)inemia due to a deficiency in cystathionine b-synthase activity, tyrosinemia type I, or liver disease (Mudd et al., 1995a). During the screening, it has been noted that despite the absence of these conditions, some infants have abnormally high plasma methionine levels. Liver biopsies from a few of these hypermethioninemic individuals showed that they were deficient in hepatic methionine adenosyltransferase (MAT) I/III activity (Gaull & Tallan, 1974; Finkelstein et * Corresponding author. Tel.: 301-496-1094; fax: 301-402-7784. E-mail address: [email protected] (J.Y. Chou)

1 2 3 5 6 7 8 8

al., 1975; Gout et al., 1977; Guizar-Vazquez et al., 1980; Gaull et al., 1981; Uetsuji, 1986; Gahl et al., 1987); the MAT (MAT II) activity in their erythrocytes, lymphocytes, and fibroblasts was normal. With the exception of two hypermethioninemic patients who had developed abnormal neurological symptoms, including brain demyelination (Surtees et al., 1991; Mudd et al., 1995b), most individuals generally are free of major clinical manifestations (Gaull & Tallan, 1974; Finkelstein et al., 1975; Gout et al., 1977; Guizar-Vazquez et al., 1980; Gaull et al., 1981; Congdon et al., 1981; Tsuchiyama et al., 1982; Uetsuji, 1986; Gahl et al., 1987; Blom et al., 1992). A few patients develop unusual breath odor due to the presence of dimethylsulfide (Gahl et al., 1987; Mudd et al., 1995a). Hepatic MAT deficiency, therefore, is characterized by isolated hyperme-

0163-7258/99/$ – see front matter Published 1999 by Elsevier Science Inc. All rights reserved. PII: S0163-7258(99)00 0 4 7 - 9

2

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

Fig. 1. Synthesis of AdoMet from ATP and methionine catalyzed by MAT.

thioninemia in the absence of cystathionine b-synthase deficiency, tyrosinemia type I, or liver disease (Mudd et al., 1995a). The structure and function of MAT from different species (Kotb & Geller, 1993) and the regulation of the methylation cycle and its alteration during liver dysfunction (Mato et al., 1997) have been reviewed recently. This review focuses primarily on the molecular genetics of hepatic MAT deficiency. 2. Methionine adenosyltransferase and S-adenosylmethionine MAT catalyzes the biosynthesis of S-adenosylmethionine (AdoMet) from methionine and ATP (Fig. 1) (Cantoni, 1953). AdoMet plays a pivotal role in metabolism, participating in the transmethylation and trans-sulfuration pathways and the biosynthesis of polyamines (reviewed in Tabor & Tabor, 1984; Kotb & Geller, 1993; Mudd et al., 1995a; Chiang et al., 1996). The transfer of a methyl group from AdoMet to a methyl acceptor results in the conversion of AdoMet to S-adenoyslhomocysteine, which in turn is hydrolyzed to adenosine and homocysteine (Fig. 2). Homocysteine can then either undergo methylation to methionine and re-enter the transmethylation cycle or participate in the trans-sulfuration pathway for the biosynthesis of compounds such as cysteine and glutathione. AdoMet can also undergo decarboxylation to generate the propylamine donor for the biosynthesis of polyamines. The polyamines are essential for vital cellular functions, such as growth and differentiation. They also act as intracellular messengers to regulate free cytosolic Ca21 levels (reviewed in Janne et al., 1991). The overall reaction of MAT is composed of two sequential steps, AdoMet formation and the subsequent tri-

polyphosphate hydrolysis that occurs prior to release of AdoMet from the enzyme (Fig. 1). cDNAs encoding MAT have been isolated and characterized from many species, including Escherichia coli, yeast, plant, human, rat, and mouse (reviewed in Kotb & Geller, 1993). At least two MAT genes are present in all species studied so far, and an extensive sequence conservation is observed across all species. Taken together with the central role that MAT is known to play in metabolism, it is evident that MAT is an essential enzyme for life. Three MAT isozymes, I, II, and III, exist in mammals. The nomenclature of the mammalian MAT isozymes was

Fig. 2. The metabolism of methionine.

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

3

Table 1 Consensus nomenclature of mammalian MAT genes and gene products MAT isozyme

Catalytic subunit

Gene encoding MAT catalytic subunit

Regulatory subunit

Gene encoding MAT regulatory subunit

Subunit composition

MAT I MAT II MAT III

a1 a2 a1

MAT1A MAT2A MAT1A

b

MAT2B

(a1)4 (a2,a29)xby (a1)2

Reproduced from Kotb et al. (1997), with permission of the authors and the copyright holder, Elsevier Trends Journals, Cambridge.

based on increasing order of hydrophobicity when eluted from phenyl-Sepharose columns. The catalytic subunits of mammalian MAT isozymes are encoded by two distinct genes, MAT1A and MAT2A. While MAT I and III are different oligomeric states of the same a1-subunit, MAT II consists of a2 catalytic and b-regulatory subunits in an unknown stoichiometry (Kotb & Geller, 1993). The gene encoding the b-subunit has not been characterized. Consensus nomenclature of the mammalian MAT genes and gene products has been recommended and adopted (Table 1) (Kotb et al., 1997). MAT I, II, and III have distinct kinetic properties for methionine and ATP, and are differentially regulated by dimethylsulfoxide and AdoMet (reviewed in Tabor & Tabor, 1984; Kotb & Geller, 1993). MAT III, which is the major hepatic isozyme, is activated by both dimethylsulfoxide and AdoMet. Both MAT I and III activities are markedly reduced by sulfhydryl reagents. MAT II is found in fetal liver (and to a lesser extent in adult liver), as well as in kidney, brain, testis, and lymphocytes (reviewed in Kotb & Geller, 1993). MAT II activity is inhibited by dimethylsulfoxide and AdoMet, but is resistant to inhibition by sulfhydryl reagents. 3. The human methionine adenosyltransferase genes and the encoded isozymes The two human MAT (hMAT) genes, hMAT1A and hMAT2A, and the corresponding cDNAs have been isolated and characterized (Horikawa & Tsukada, 1991, 1992; Alva-

rez et al., 1993; Ubagai et al., 1995; De La Rosa et al., 1995; Mao et al., 1998). The hMAT1A gene is expressed primarily in the liver and encodes the a1-subunit of the tetrameric hMAT I [(a1)4] and dimeric hMAT III [(a1)2] isozymes, usually referred to together as MAT I/III. The hMAT1A gene comprises 9 exons spanning approximately 20 kb and located on chromosome 10q22 (Ubagai et al., 1995) (Fig. 3). The sizes of the exons are I, 162 bp, including 91 bp coding and 71 bp 59-untranslated sequences; II, 78 bp; III, 123 bp; IV, 113 bp; V, 144 bp; VI, 219 bp; VII, 183 bp; VIII, 134 bp; and IX, 2061 bp, including 103 bp coding and 1958 bp 39-untranslated sequences. Southern-blot analyses of human genomic DNA showed that exon sequences were contained within two BamHI (.20 and 4.5 kb) or two EcoRI (14 and 3.8 kb) fragments, as predicted from the MAT gene, suggesting that hMAT1A is a single copy gene (Ubagai et al., 1995). The hMAT2A gene that encodes the a2-subunit maps to chromosome 2p11.2 (De La Rosa et al., 1995). The structural organization of the hMAT2A gene has not been characterized. Recently, it was shown that the exon structure and the insertion sites of the corresponding introns of the rat MAT2A gene (Hiroki et al., 1997) are similar to that of the mouse (Sakata et al., 1993) and human (Ubagai et al., 1995) MAT1A genes, suggesting that the hMAT2A gene is similarly organized. The hMATa1- and hMATa2-subunits are predicted to be polypeptides of 395 amino acids (Horikawa & Tsukada, 1991, 1992; Alvarez et al., 1993; Ubagai et al., 1995), sharing 84% sequence identity (Fig. 4). Recently, the crystal

Fig. 3. The structural organization and chromosomal location of the hMAT1A transcription unit. The exon coding regions are indicated by filled boxes and the untranslated regions, by open boxes. The location of the hMAT1A gene is indicated by an arrow. B, BamHI; R, EcoRI.

Fig. 4. The aligned amino acid sequences of hMATa1, hMATa2, and E. coli MAT subunits. The amino acid sequence of hMATa1 is shown in its entirety. Exact matches are denoted by dashes, and asterisks are used when amino acids are skipped to allow for the best alignment. Amino acids contributing to the active center of MAT are in bold and indicated as #. Amino acids mutated in hepatic MAT-deficient individuals are shaded and indicated as •. D134, an amino acid in the conserved ATP binding motif (GXGDXG), is included in the active center of MAT because computer modeling predicts that it forms an inter-subunit salt bridge with K181.

4 J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

5

Fig. 5. Interaction of ADP, Pi, and metal ions in the MAT active site. Residues from subunits A and B are distinguished by asterisks.

structure for the tetrameric E. coli MAT (Markham et al., 1984) has been determined (Takusagawa et al., 1996a, 1996b), which predicts that the active site of MAT is at the interface of two identical subunits. The amino acids predicted to participate in E. coli MAT catalysis are well conserved in mammalian MATs. The corresponding amino acids in hMATa1 include His-29, Asp-31, Glu-57, Gln-113, Asp-134, Lys-181, Arg-264, Lys-265, Ser-283, Lys-285, Lys-289, and Asp-291 (Fig. 5). Thus, the crystal structure of the E. coli MAT can be used as the prototype to delineate the amino acid residues that contribute to MAT catalysis in mammals and to increase our understanding of the pathogenesis of hepatic MAT deficiency.

clinical symptoms, including generalized hypotonia, with death at 8 months (Guizar-Vazquez et al., 1980); mental retardation (Mudd et al., 1995b); and brain demyelination (Surtees et al., 1991). With the cloning of the hMAT1A gene (Ubagai et al., 1995), gene-based diagnostic tests for this disorder became possible, and are being developed. Mutations can now be accurately and rapidly diagnosed by various molecular methods, including single-strand conformation polymorphism analysis and DNA sequencing. To date, mutations have been identified in the hMAT1A genes of 11 individuals and 3 pedigrees with the isolated hypermethioninemic phenotype (Table 2) (Ubagai et al., 1995; Chamberlin et al., 1996, 1997; Nagao & Oyanagi,

4. The molecular basis of hepatic methionine adenosyltransferase deficiency

Table 2 Hepatic MAT-deficient patients and pedigrees

Hepatic MAT deficiency was first identified by assay of MAT activity in a liver biopsy sample of an infant with isolated persistent hypermethioninemia by Gaull and Tallan (1974). Hepatic MAT assays were subsequently used to identify additional hepatic MAT-deficient individuals with high plasma methionine levels (Finkelstein et al., 1975; Gout et al., 1977; Guizar-Vazquez et al., 1980; Gaull et al., 1981; Uetsuji, 1986; Gahl et al., 1987). However, many of the isolated hypermethioninemic individuals with plasma methionine concentrations ranging from 100 to 2500 mM (versus a reference methionine level of 35–45 mM) have been identified (Mudd et al., 1995b); they were not positively diagnosed by MAT assays. Although most of these individuals are clinically well, it has been noted that a few isolated hypermethioninemic patients suffer from severe

Patienta/pedigree

Ageb

Reference

G1 (F) G2 (M) G3 (F) G4 (F) C (F) 3 (F) 8 (F) 9 (M) 10 (M) 13 (F) P (M) Pedigree G Pedigree C Pedigree J

6 0.3 2 0.7 11 4 14 1.8 5.9 2.6 43

Gaull et al., 1981 Gaull et al., 1981 Gaull et al., 1981 Gaull et al., 1981 Surtees et al., 1991 Mudd et al., 1995b Mudd et al., 1995b Mudd et al., 1995b Mudd et al., 1995b Mudd et al., 1995b Hazelwood et al., 1998 Chamberlin et al., 1997 Chamberlin et al., 1997 Nagao and Oyanagi, 1997

a b

Patient numbers are taken from the cited references. Age (years) at last report.

6

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

827insG, and 1043delTG, totally abolished MAT activity. Taken together, these studies demonstrate that lesions in the hMAT1A gene cause hepatic MAT deficiency, firmly establishing the molecular basis of this disorder.

Table 3 Mutations identified in the hMAT1A genes of hepatic MAT-deficient individuals Nucleotides/amino acids

Mutations

Reference

164C→A/A55D 595C→T/R199C 914T→C/L305P 966T→G/I322M 1068G→A/R356Q 1070C→T/P357L 1132G→A/G378S 539insTG/185X

Missense Missense Missense Missense Missense Missense Missense Insertion

827insG/351X 1043delTG/350X 791G→A/R264H

Insertion Deletion Missense

Ubagai et al., 1995 Chamberlin et al., 1996 Ubagai et al., 1995 Ubagai et al., 1995 Chamberlin et al., 1996 Ubagai et al., 1995 Chamberlin et al., 1996 Chamberlin et al., 1996; Hazelwood et al., 1998 Chamberlin et al., 1996 Chamberlin et al., 1996 Chamberlin et al., 1997; Nagao and Oyanagi, 1997

1997; Hazelwood et al., 1998). A total of 11 separate mutations have been identified in the coding sequence of the hMAT1A genes of 7 homozygotes, 4 compound heterozygotes, and the 3 pedigrees (Tables 3 and 4). These include 8 missense and 3 insertion/deletion mutations (Table 3). All 8 missense mutations reduced enzymatic activity when assayed following transient expression of appropriately mutagenized cDNAs; none completely abolished MAT activity (Ubagai et al., 1995; Chamberlin et al., 1996, 1997) (Table 4). However, the 3 insertion/deletion mutations, 539insTG,

4.1. The hypermethioninemic phenotype can be transmitted both as autosomal recessive and dominant traits As determined by segregation and mutation analysis, the hypermethioninemic phenotype in the majority of hepatic MAT-deficient individuals is transmitted as an autosomal recessive trait (Ubagai et al., 1995; Chamberlin et al., 1996), as demonstrated in the seven homozygous and four compound heterozygous individuals (Table 4). A dominantly inherited form of isolated hypermethioninemia has been reported in two families by genetic analysis (Blom et al., 1992; Mudd et al., 1995b). When these two families were examined, a single 791G→A/R264H mutation was detected in one hMAT1A allele of both families (Chamberlin et al., 1997). This dominant inherited mutation was later identified in another hypermethioninemic pedigree in Japan (Nagao & Oyanagi, 1997). The enzymatically active MAT is a MATa1 oligomer (Cabrero et al., 1987). Therefore, the pattern of dominant inheritance observed in the 791G→A/R264H mutation in the hMAT1A gene suggests that R264 may be involved in subunit dimerization. It also suggests that heterodimers formed between a wild-type and a R264H mutant hMATa1-subunit

Table 4 Mutations identified in the MAT1A gene that are transmitted as an autosomal recessive trait Patient

Nucleotides/amino acids

G1

966T→G/I322M 966T→G/I322M 914T→C/L305P 966T→G/I322M 164C→A/A55D 1070C→T/P357L 1068G→A/R356Q 1132G→A/G378S 827insG/351X 827insG/351X 539insTG/185X 539insTG/185X 1043delTG/350X 1043delTG/350X 539insTG/185X 595C→T/R199C 595C→T/R199C 595C→T/R199C 595C→T/R199C 595C→Τ/Ρ199Χ 539insTG/185X 539insTG/185X

G2 G3 G4 C 3 8 9 10 13 P a

Plasmaa methionine

MAT activityb

Comments

Reference

0.24 6 0.04 (11.4)

Normal

Ubagai et al., 1995

Normal

Ubagai et al., 1995

Normal

Ubagai et al., 1995

Normal

Chamberlin et al., 1996

600–1400

0.34 6 0.01 (16.1) 0.24 6 0.04 (11.4) 0.14 6 0.06 (6.6) 0.47 6 0.08 (22.3) 6.43 6 0.44 (53.1) 0.19 6 0.01 (0.17) 0.0

Chamberlin et al., 1996

686–2541

0.0

Dyspraxia, demyelination Calcification of basal ganglia Normal

1114–1629

0.0

127 58.3 102 254–400

759–1467

Chamberlin et al., 1996

Retarded dystonia Demyelination Normal

Chamberlin et al., 1996 Chamberlin et al., 1996

Normal

Chamberlin et al., 1996

484–742

0.0 0.92 6 0.02 (11.1) 0.92 6 0.02 (11.1)

451–758

0.92 6 0.02 (11.1)

Normal

Chamberlin et al., 1996

686–2541

N.D.

Normal

Hazelwood et al., 1998

Plasma methionine (micromolar) levels previously published in references listed in Table 1. MAT activity (nmol/min/mg protein) was presented as the mean 6 SEM. Mock MAT activity has been subtracted from each sample. Data from Ubagai et al. (1995) were determined from COS-1 expressed wild-type and mutant hMAT1A cDNA. MAT activity in wild-type cDNA and mock-transfected COS-1 cultures were 2.38 6 0.23 and 0.27 6 0.01, respectively. Data from Chamberlin et al. (1996) were determined from bacterially expressed wild-type and mutant MATA1 cDNA. MAT activity in wild-type cDNA transformed and mock bacterial cultures were 16.55 6 0.56 and 0.17 6 0.01, respectively. Numbers in parentheses represent percent of wild-type activity. N.D., not determined. b

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

7

Fig. 6. Western blot analysis and enzymatic activity of bacterially expressed wild-type or mutant hMAT1A cDNAs. Bacterial extracts were incubated in the absence (–) or presence (1) of a chemical cross-linker Bis-(sulfosuccinimidyl)-suberate. Numbers in parentheses represent percent of wild-type activity. Modified from Chamberlin et al. (1997).

is enzymatically inactive. Three lines of evidence supported this conclusion (Chamberlin et al., 1997): (1) transient expression assays showed that the R264H mutant had less than 1% of the wild-type MAT activity (Fig. 6); (2) while wild-type hMATa1-subunits formed dimers readily, the R264H mutant subunit failed to dimerize (Fig. 6); and (3) when wild-type R264 and mutant R264H cDNAs were cotransfected into COS-1 cells, enzymatically inactive hMATa heterodimers were formed (Chamberlin et al., 1997). Based on the crystal structure of E. coli MAT, R264 is predicted to be involved in salt-bridge formation (Fig. 5), which is essential for subunit dimerization and optimal MAT activity (Takusagawa et al., 1996a, 1996b). Among the eight amino acids altered by missense mutations identified in the hMAT1A genes of hepatic MAT-deficient individuals, residue 264 is the only amino acid that is predicted to be at the active center of the enzyme (Fig. 4). Mutational and transient expression studies have been employed to examine the structural requirement of residue 264 in MAT catalysis by Chamberlin et al. (1997). R264 was replaced with the structurally dissimilar His, Leu, Asp, or Glu and structurally similar Lys, and the mutant constructs were examined for MAT activity and the ability to form dimers. R264H, R264L, R264D, and R264E mutants had markedly reduced MAT activity, and they were unable to form homodimers (Fig. 6). On the other hand, the R264K mutant a1-subunit was able to form dimers that retained significant (20.4% of wild-type) MAT activity. These studies indicate that a positive charge at residue 264 is necessary, but not sufficient, for full enzymatic activity.

4.2. The phenotype of a null hMAT1A mutation Among the patients characterized to date, four patients (C, 3, 8, and P) are homozygous for hMAT1A mutations that yield truncated a1-subunits devoid of enzymatic activity (Table 4). These four patients have unusually high plasma methionine concentrations (Table 4), and it appears that plasma methionine concentrations are inversely correlated to hepatic MAT activity in hypermethioninemic individuals (Chamberlin et al., 1996). The wild-type hMATa1-subunit is a polypeptide of 395 amino acids (Horikawa & Tsukada, 1991; Alvarez et al., 1993; Ubagai et al., 1995), and the mutant subunits in patients C, 3, 8, and P are predicted to be polypeptides of 350, 184, 349, and 184 amino acids, respectively (Chamberlin et al., 1996; Hazelwood et al., 1998). Patients C (Surtees et al., 1991) and 8 (Mudd et al., 1995b) manifested brain demyelination at 11 years of age, and it has been speculated that this demyelination is caused by the lack of synthesis of AdoMet and a resulting lack of methylated products, which include phosphatidylcholine and creatine (Chamberlin et al., 1996). Phosphatidylcholine, along with sphingomyelin, makes up 28–34% of the composition of the myelin in the neural sheath (Norton & Cammer, 1984). Brain creatine is also important to neuronal structure, with deficiencies showing an association with severe neurological manifestations (Stockler et al., 1996). The link between MAT activity, myelination, and neural functions is interesting, and should be explored further. However, patient 3 (4 years of age) and patient P (43 years of age) are clinically well, despite the

8

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9

fact that both are homozygous for the 539insTG mutation, which is predicted to encode a severely truncated MATa1subunit of 184 amino acids having no enzymatic activity. At present, one cannot rule out yet the possibility that the apparent correlation between the absence of hepatic MAT activity and the brain demyelination phenotype reflects a simple coincidence. It is also possible that the loss of MAT activity predisposes patients to demyelination and that clinical outcome depends on their genetic background. An alternative explanation proposed by Hazelwood et al. (1998) suggests that patients 3 and P are clinically well because their mutant MATa1-subunit of 184 amino acids cannot heterodimerize with the MATa2-subunit, and thus cannot adversely inhibit hepatic MAT II activity. This severely truncated MATa1 mutant subunit lacks 6 (R264, K265, S283, K285, D291, and K289) of the 12 amino acids predicted to be involved in MAT catalysis (Fig. 4). As noted in Section 4.1, R264 plays a role in subunit dimerization that is essential for enzymatic activity (Takusagawa et al., 1996a, 1996b; Chamberlin et al., 1997). The hMATa1- and hMATa2-subunits share 84% sequence identity, including the 12 amino acids predicted to contribute to the active center of MAT (Fig. 4). Thus, it is possible that patients C and 8 suffer from brain demyelination because their mutant MATa1-subunits of 350 and 349 amino acids can dimerize with the hepatic MATa2-subunit, forming an enzymatically inactive heterodimer. Brain demyelination recently has been identified in another isolated hypermethioninemic individual (M. E. Chamberlin, S. H. Mudd, & J. Y. Chou, unpublished results). Therefore, it is possible that the 7% MAT activity in adult liver contributed by the MAT II isozyme (Hazelwood et al., 1998) is essential for the maintenance of normal myelination in the brain. 5. Conclusion Molecular genetic evidence has unequivocally demonstrated that lesions in the hMAT1A gene that abolish or greatly reduce enzymatic activity cause hepatic MAT deficiency, resulting in the persistent hypermethioninemic phenotype. However, the relationship between hepatic MAT activity and brain myelination remains unclear. To address this question, the hMAT1A gene should be characterized in additional individuals who have usually high plasma methionine levels (.1000 mM). If a complete lack of hepatic MAT activity leads to brain demyelination, this information should guide physicians to implement appropriate therapies that could prevent neurological manifestations. References Alvarez, L., Corrales, F., Martin-Duce, A., & Mato, J. M. (1993). Characterization of a full-length cDNA encoding human liver S-adenosylmethionine synthetase: tissue-specific gene expression and mRNA levels in hepatopathies. Biochem J 293, 481–486. Blom, H. J., Davidson, A. J., Finklestein, J. D., Luder, A. S., Bernardini, I.,

Martin, J. J., Tangerman, A., Trijbels, J. M. F., Mudd, S. H., Goodman, S. I., & Gahl, W. A. (1992). Persistent hypermethioninemia with a dominant inheritance. J Inherit Metab Dis 15, 188–197. Cabrero, C., Puerta, J., & Alemany, S. (1987). Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver. Eur J Biochem 170, 299–304. Cantoni, G. L. (1953). S-Adenosylmethionine: a new intermediate formed enzymatically from L-methionine and adenosine triphosphate J Biol Chem 204, 403–416. Chamberlin, M. E., Ubagai, T., Mudd, S. H., Wilson, W. G., Leonard, J. V., & Chou, J. Y. (1996). Demyelination of the brain is associated with methionine adenosyltransferase I/III deficiency. J Clin Invest 98, 1021–1027. Chamberlin, M. E., Ubagai, T., Mudd, S. H., Levy, H. L., & Chou J. Y. (1997). Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene. Am J Hum Genet 60, 540–546. Chiang, P. K., Gordon, R. K., Tal, J., Zeng, G. C., Doctor, B. P., Pardhasaradhi, K., & McCann, P. P. (1996). S-Adenosylmethionine and methylation. FASEB J 10, 471–480. Congdon, P. J., Haigh, D., Smith, R., Green, A., & Pollitt, R. J. (1981). Hypermethioninemia and 3-hydroxyisobutyric aciduria in an apparently healthy baby. J Inherit Metab Dis 4, 79–80. De La Rosa, J., Ostrowski, J., Hryniewicz, M. M., Kredich, N. M., Kotb, M., LeGros, H. L., Jr., Valentine, M., & Geller, A. M. (1995). Chromosomal localization and catalytic properties of the recombinant a subunit of human lymphocyte methionine adenosyltransferase. J Biol Chem 270, 21860–21868. Finkelstein, J. D., Kyle, W. E., & Martin, J. J. (1975). Abnormal methionine adenosyltransferase in hypermethioninemia. Biochem Biophys Res Commun 66, 1491–1497. Gahl, W. A., Finkelstein, J. D., Mullen, K. D., Bernardini, I., Martin, J. J., Backlund, P., Ishak, K. G., Hoofnagle, J. H., & Mudd, S. H. (1987). Hepatic methionine adenosyltransferase deficiency in a 31-year-old man. Am J Hum Genet 40, 39–49. Gaull, G. E., & Tallan, H. H. (1974). Methionine adenosyltransferase deficiency: new enzymatic defect associated with hypermethioninemia. Science 186, 59–60. Gaull, G. E., Tallan, H. H., Lansdale D., Przyrembel, H., Schaffner, F., & von Bassewitz, D. B. (1981). Hypermethioninemia associated with methionine adenosyltransferase deficiency: clinical, morphologic, and biochemical observations on four patients. J Pediatr 98, 734–741. Gout, J.-P., Serre, J.-C., Dieterlen, M., Antener, I., Frappat, P., Bost, M., & Beaudoing, A. (1977). Une nouvelle cause d’hypermethioninemie de l’enfant: le deficit en S-adenosyl-methionine-synthetase. Arch Fr Pediatr 34, 416–423. Guizar-Vazquez, J., Sanchez-Aguilar, G., Velazquez, A., Fragoso, R., Rostenbery, I., & Alejandre, I. (1980). Hipermethioninemia. A proposito de un caso en un matrimonio consanguineo. Bol Med Hosp Infant Mex 37, 1237–1244. Hazelwood, S., Bernardini, I., Shotelersuk, V., Tangerman, A., Guo, J., Mudd, H., & Gahl, W. A. (1998). Normal brain myelination in a patient homozygous for a mutation that encodes a severely truncated methionine adenosyltransferase I/III. Am J Med Genet 75, 395–400. Hiroki, T., Horikawa, S., & Tsukada, K. (1997). Structure of the rat methionine adenosyltransferase 2A gene and its promoter. Eur J Biochem 250, 653–660. Horikawa, S., & Tsukada, K. (1991). Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase. Biochem Int 25, 81–90. Horikawa, S., & Tsukada, K. (1992). Molecular cloning and developmental expression of a human kidney S-adenosylmethionine synthetase. FEBS Lett 312, 37–41. Janne, J., Alhonen, L., & Leinonen, P. (1991). Polyamines: from molecular biology to clinical applications. Ann Med 23, 241–259. Kotb, M., & Geller, A. M. (1993). Methionine adenosyltransferase: structure and function. Pharmacol Ther 59, 125–143.

J.Y. Chou / Pharmacology & Therapeutics 85 (2000) 1–9 Kotb, M., Mudd, S. H., Mato, J. M., Geller, A. M., Kredich, N. M., Chou, J. Y., & Cantoni, G. L. (1997). Consensus nomenclature for the mammalian methionine adenosyltransferase genes and gene products. Trends Genet 13, 51–52. Mao, Z., Liu, S., Cai, J., Huang, Z. Z., & Lu, S. C. (1998). Cloning and functional characterization of the 59-flanking region in human methionine adenosyltransferase 2A gene. Biochem Biophys Res Commun 248, 479–484. Markham, G. D., DeParasis, J., & Gatmaitan, J. (1984). The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli. J Biol Chem 259, 14505–14507. Mato, J. M., Alvarez, L., Ortiz, P., & Pajares, M. A. (1997). S-Adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol Ther 73, 265–280. Mudd, S. H., Levy, H. L., & Skovby, F. (1995a). Disorders of transsulfuration. In C. R. Scriver, A. L. Beaudet, W. S. Sly, & D. Valle (Eds.), The Molecular and Metabolic Basis of Inherited Diseases, 7th edn. (pp. 1279–1327). New York: McGraw-Hill. Mudd, S. H., Levy, H. L., Tangerman, A., Boujet, C., Buist, N., DavidsonMundt, A., Hudgins, L., Oyanagi, K., Nagao, M., & Wilson, W. G. (1995b). Isolated persistent hypermethioninemia. Am J Hum Genet 57, 882–892. Nagao, M., & Oyanagi, K. (1997). Genetic analysis of isolated persistent hypermethioninemia with dominant inheritance. Acta Pediatr Jpn 39, 601–606. Norton, W. T., & Cammer, W. (1984). Isolation and characterization of myelin. In P. Morell (Ed.), Myelin (pp. 147–195). New York: Plenum Press. Sakata, S. F., Shelly, L. L., Ruppert, S., Schutz, G., & Chou, J. Y. (1993).

9

Cloning and expression of murine S-adenosylmethionine synthetase. J Biol Chem 268, 13978–13986. Stockler, S., Isbrandt, D., Hanefeld, F., Schmidt, B., & von Figura, K. (1996). Guanidinoacetate methyltransferase deficiency: the first inborn error of creatine metabolism in man. Am J Hum Genet 58, 914–922. Surtees, R., Leonard, J., & Austin, S. (1991). Association of demyelination with deficiency of cerebrospinal-fluid S-adenosylmethionine in inborn errors of methyl-transfer pathway. Lancet 338, 1550–1554. Tabor, C. W., & Tabor, H. (1984). Methionine adenosyltransferase (S-adenosylmethionine synthetase) and S-adenosylmethionine decarboxylase. Adv Enzymol 56, 251–282. Takusagawa, F., Kamitori, S., & Markham, G. D. (1996a). Structure and function of S-adenosylmethionine synthetase: crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi,at 2.8Å resolution. Biochemistry 35, 2586–2596. Takusagawa, F., Kamitori, S., Misaki, S. & Markham, G. D. (1996b). Crystal structure of S-adenosylmethionine synthetase. J Biol Chem 271, 136–147. Tsuchiyama, A., Oyanagi, K., Nakata, F., Uetsuji, N., Tsugawa, S., Nakao, T., & Mori, M. (1982). A new type of hypermethioninemia in neonates. Tokohu J Exp Med 138, 281–288. Ubagai T., Lei, K.-J., Huang, S., Mudd, S. H., Levy, H. L., & Chou, J. Y. (1995). Molecular mechanisms of an inborn error of methionine pathway: methionine adenosyltransferase deficiency. J Clin Invest 96, 1943–1947. Uetsuji, N. (1986). Genetical and biochemical studies in patients with congenital hypermethioninemia. J Clin Pediatr (Sapporo) 34, 167–179.