Methionine synthase

Methionine synthase (MetH) of E. coli represents the most thoroughly studied B12-dependent methyl transferase and is one of the essential roles of B12 in mammalian metabolism [125,153,154]. It is a modular enzyme containing separate binding domains for homocysteine, N -methyltetrahydrofolate, S-adenosyl-methionine (SAM) and the Bi2-cofactor [125,153-155]. The B12-binding domain in its different oxidation states must interact punctually and specifically with each of the other three domains The Co(I) form with the N -methyltetrahydrofolate binding domain, the Co(II) form with the SAM binding domain, and the CH3 - Co (III) form with the homocysteine binding domain [153,155]. 

MetH catalyzes the methylation of the bound and reduced cob(I)alamin cofactor by (N -protonated) N -methyltetrahydrofolate to give enzyme-bound methylcobalamin (3) in a base-off/His-on form (see later) [125,153-155]. The methyl-Co(III)corrinoid is demethylated by homocysteine, whose sulfur is activated and deprotonated due to the coordination to a zinc ion (held by three cysteine residues) of the homocysteine binding domain [164] (see Fig. 15). The two methyl-transfer reactions occur in a sequential mechanism [124,125,153,154]. Intermittently, the bound Cob(I)alamin (40 ) is oxidized to enzymatically inactive cob(II)alamin (23) and requires reactivation by reductive methylation with SAM and a flavodoxin as a reducing agent [125,153-155,165]. 

The x-ray crystal analysis of the B -binding domain of MetH provided the first insight into the three-dimensional structure of a Bn-binding protein [17, 

The crystallographic results on the structure of MetH and the finding of the base-off/His-on binding of the cofactor in a Bi2-dependent methyl transferase were consistent with earlier ESR spectroscopic evidence for histidine binding to the cobalt center of p-cresolyl-cobamide (52) in the aceto-gen Sporomusa ovata [145,169]. Various other B -dependent methyltrans-ferases are indicated to have either a base-off/His-on bound methyl-Co(III)-corrinoid, or even a methyl-corrinoid cofactor in base-off form (where His-coordination is absent) [156]. 

In a catalytic cycle of B -dependent methyl transferases the corrinoid is indicated to cycle between a methyl-Co(III)-corrin and a Co(I)-corrin [125,126, 153,155]. The changing between the hexacoordinate methyl-Co(III)-form and (presumably) tetracoordinate Co(I)-form is accompanied by constitu-tional/conformational changes, which are highly likely to provide a means for controlling the organometallic reactivity of the bound cofactor [170], sub- 

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Fohc acid is a precursor of several important enzyme cofactors required for the synthesis of nucleic acids (qv) and the metaboHsm of certain amino acids. Fohc acid deficiency results in an inabiUty to produce deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and certain proteins (qv). Megaloblastic anemia is a common symptom of folate deficiency owing to rapid red blood cell turnover and the high metaboHc requirement of hematopoietic tissue. One of the clinical signs of acute folate deficiency includes a red and painhil tongue. Vitamin B 2 folate share a common metaboHc pathway, the methionine synthase reaction. Therefore a differential diagnosis is required to measure foHc acid deficiency because both foHc acid and vitamin B 2 deficiency cause... 

N5-Methyltetrahydrofolate homocysteine methyl-transferase (= methionine synthase). This reaction is essential to restore tetrahydrofolate from N5-methyltetrahydrofolate (Fig. 2). 

Methylmalonyl CoA mutase, leucine aminomutase, and methionine synthase (Figure 45-14) are vitamin Bj2-dependent enzymes. Methylmalonyl CoA is formed as an intermediate in the catabolism of valine and by the carboxylation of propionyl CoA arising in the catabolism of isoleucine, cholesterol, and, rarely, fatty acids with an odd number of carbon atoms—or directly from propionate, a major product of microbial fer-... 

Figure 45-14. Homocysteinuria and the folate trap. Vitamin 6,2 deficiency leads to inhibition of methionine synthase activity causing homocysteinuria and the trapping of folate as methyltetrahydrofolate.

When acting as a methyl donor, 5-adenosylmethionine forms homocysteine, which may be remethylated by methyltetrahydrofolate catalyzed by methionine synthase, a vitamin Bj2-dependent enzyme (Figure 45-14). The reduction of methylene-tetrahydrofolate to methyltetrahydrofolate is irreversible, and since the major source of tetrahydrofolate for tissues is methyl-tetrahydrofolate, the role of methionine synthase is vital and provides a link between the functions of folate and vitamin B,2. Impairment of methionine synthase in Bj2 deficiency results in the accumulation of methyl-tetrahydrofolate—the folate trap. There is therefore functional deficiency of folate secondary to the deficiency of vitamin B,2. 

In mammals and in the majority of bacteria, cobalamin regulates DNA synthesis indirectly through its effect on a step in folate metabolism, catalyzing the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate via two methyl transfer reactions. This cytoplasmic reaction is catalyzed by methionine synthase (5-methyltetrahydrofolate-homocysteine methyl-transferase), which requires methyl cobalamin (MeCbl) (253), one of the two known coenzyme forms of the complex, as its cofactor. 5 -Deoxyadenosyl cobalamin (AdoCbl) (254), the other coenzyme form of cobalamin, occurs within mitochondria. This compound is a cofactor for the enzyme methylmalonyl-CoA mutase, which is responsible for the conversion of T-methylmalonyl CoA to succinyl CoA. This reaction is involved in the metabolism of odd chain fatty acids via propionic acid, as well as amino acids isoleucine, methionine, threonine, and valine. 

In contrast with the role of cofactor B12 in methionine synthase (methyl group transfer to a thiol), functional Bi2 model complexes have provided a formidable challenge. Several oxime alkyl-cobalt (structural) B12 models when reacted with arene- and alkanethiolates lead only to... 

Riordan and co-workers have examined zinc complexes of pyrazolyl-bis[(methylthio)methyl]-borate ligands as models for methionine synthase.547, 69 The ligand coordinates in a face-capping fashion with the desired NS2 donor set. 

Flavin Mononucleotide (FMN) Methionine synthase reductase, Chorismate synthase... 

Zinc is the active metal in the largest group of metalloproteins found in the nature. Recently a new class of zinc enzymes with a sulfur-rich environment has emerged the thiolate-alkylating enzimes, the most prominent of which is the cobalamine-independent methionine synthase.126 For these reasons several monothiolate zinc complexes have been prepared for the modelling of these enzymes with different N2S as (13),127 130 N20,13° 132 N3,132,133 S3,134 tripod ligands, or with Cd because of the favourable spectroscopic properties with an S3 tripod ligand.135... 

Methionine synthase deficiency (cobalamin-E disease) produces homocystinuria without methylmalonic aciduria 677 Cobalamin-c disease remethylation of homocysteine to methionine also requires an activated form of vitamin B12 677 Hereditary folate malabsorption presents with megaloblastic anemia, seizures and neurological deterioration 678... 

In cobalamin-E (cblE) disease there is a failure of methyl-B12 to bind to methionine synthase. It is not known if this reflects a primary defect of methionine synthase or the absence of a separate enzyme activity. Patients manifest megaloblastic changes with a pancytopenia, homocystinuria and hypomethioninemia. There is no methylmalonic aciduria. Patients usually become clinically manifest during infancy with vomiting, developmental retardation and lethargy. They respond well to injections of hydroxocobalamin. 

SMM synthesis is mediated by the enzyme methionine S-methyltransferase (MMT) through the essentially irreversible, AdoMet-mediated methylation of methionine.48"5 Both MMT and SMM are unique to plants 48,50 The opposite reaction, in which SMM is used to methylate homocysteine to yield two molecules of methionine, is catalyzed by the enzyme homocysteine S-methyltransferase (HMT).48 Unlike MMT, HMTs also occur in bacteria, yeast, and mammals, enabling them to catabolize SMM of plant origin, and providing an alternative to the methionine synthase reaction as a means to methylate homocysteine. Plant MMT and HMT reactions, together with those catalyzed by AdoMet synthetase and AdoHcy hydrolase, constitute the SMM cycle (Fig. 2.4).4... 

MATTHEWS, R.G., SHEPPARD, C GOULDING, C., Methylenetetrahydrofolate reductase and methionine synthase biochemistry and molecular biology, Eur. J. Pediatr., 1998,157, S54-S59. 

The best characterized B 12-dependent methyltransferases is methionine synthase (Figure 15.11) from E. coli, which catalyses the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate. During the catalytic cycle, B12 cycles between CH3-Co(in) and Co(I). However, from time to time, Co(I) undergoes oxidative inactivation to Co(II), which requires reductive activation. During this process, the methyl donor is S-adenosylmethionine (AdoMet) and the electron donor is flavodoxin (Fid) in E. coli, or methionine synthase reductase (MSR) in humans. Methionine synthase... 

Figure 15.11 Reactions catalysed by cobalamin-dependent methionine synthase. (From Banneijee and Ragsdale, 2003. Reprinted with permission from Annual Reviews.)...

Figure 15.12 The modular structure of methionine synthase. The four domains are connected by flexible hinges, which allow the CH3tetrahydrofolate-, AdoMet- or homocystein-binding domains to alternatively access the B12-binding domain. (From Bannerjee and Ragsdale, 2003. Reprinted with permission from Annual Reviews.)...

The structure of cobalamin is more complex than that of folic acid (Figure 15.2 and 15.3). At its heart is a porphyrin ring containing the metal ion cobalt at its centre. In catalytic reactions the cobalt ion forms a bond with the one-carbon group, which is then transferred from one compound to another. Vitamin B12 is the prosthetic group of only two enzymes, methylmalonyl-CoAmutase and methionine synthase. The latter enzyme is particularly important, as it is essential for the synthesis of nucleotides which indicates the importance of vitamin B12 in maintenance of good health. 

Figure 22.7 Homocysteine formation from methionine and formation of thiolactone from homocysteine. The homocysteine concentration depends upon a balance between the activities of homocysteine methyltransferase (methionine synthase) and cystathionine p-synthase. Both these enzymes require vitamin B12, so a deficiency can lead to an increase in the plasma level of homocysteine. (For details of these reactions, see Chapter 15.) Homocysteine oxidises spontaneously to form thiolactone, which can damage cell membrane.

A simple observation led to the identification of homocysteine as a risk factor for coronary heart disease. Homocysteine is an intermediate in metabolism of the amino acid methionine. Indeed, the first reaction in the catabolism of methionine involves the formation of homocysteine but it can be converted back to methionine in a reaction that is catalysed by methionine synthase (see Figure 22.7). 

Enzyme (1) is methionine synthase enzyme (2) is cystathionine P-synthase... 

In a totally different field, studies were being carried out on children who had a deficiency of methionine synthase and an impaired ability to convert homocysteine to methionine, so that they had increased blood levels of homocysteine. It was noted that these children had an increased incidence of thrombosis in cerebral and coronary arteries. This led to a study which eventually showed that an increased level of homocysteine was a risk factor for coronary artery disease in adults. Since methionine synthase requires the vitamins, folic acid and B12, for its catalytic activity, it has been suggested that an increased intake of these vitamins could encourage the conversion of homocysteine to methionine and hence decrease the plasma level of homocysteine. This is particularly the case for the elderly who are undernourished (see Chapter 15 for a discussion of nutrition in the elderly). 

This cobalamin-dependent enzyme [EC 2.1.1.13], also known as methionine synthase and tetrahydropteroyl-glutamate methyltransferase, catalyzes the reaction of 5-methyltetrahydrofolate with L-homocysteine to produce tetrahydrofolate and L-methionine. Interestingly, the bacterial enzyme is reported to require 5-adenosyl-L-methionine and FADH2. See also Tetrahydropteroyl-triglutamate Methyltransferase... 

This enzyme [EC 2.1.1.14], also known as 5-methyltet-rahydropteroyltriglutamate homocysteine 5-methyltrans-ferase and methionine synthase, catalyzes the reaction of 5-methyltetrahydropteroyltri-L-glutamate with L-homo-cysteine to produce tetrahydropteroyltri-L-glutamate and L-methionine. The reaction requires the presence of phosphate. The enzyme isolated from E. coli also requires a reducing system. See N -Methyltetrahydrofo-late. Homocysteine Methyltransferase... 

METHIONINE ADENOSYLTRANSFERASE METHIONINE y-LYASE METHIONINE SULFOXIDE REDUCTASE Methionine synthase,... 

Prolonged exposure to nitrous oxide decreases methionine synthase activity and theoretically can cause megaloblastic anemia, a potential occupational hazard for staff working in inadequately ventilated dental operating suites. 

The loss of a methyl group from AdoMet in each of the reactions yields S-ad-enosylhomocysteine (AdoHcy) and this is subsequently hydrolysed to adenosine and Hey by AdoHcy-hydrolase. Hey sits at a metabolic branch point and can be remethylated to methionine by way of two reactions. One is the 5-methyltetrahydrofo-late dependent reaction catalysed by methionine synthase, which itself is reductively methylated by cobalamin (vitamin B12) and AdoMet, requiring methionine synthase reductase. 5-Methyltetrahydrofolate is generated from 5,10-methylenetetrahydrofo-late (MTHF) by MTHF reductase. The second remethylation reaction is catalysed by betaine methyltransferase, which is restricted to the liver, kidney and brain, while methionine synthase is widely distributed. 

Fig. 2.2.1 Outline of homocysteine metabolism in man. BMT Betaine methyltransferase, cblC cobalamin defect type C, cblD cobalamin defect type D, GNMT def glycine N-methyltransferase deficiency, MAT methionine adenosyl transferase, MeCbl methylcobalamin, Met Synth methionine synthase, MTHFR methylenetetrahydrofolate reductase, SAH Hyd dc/S-adenosylhomocys-...

A large elevation of Hey in body fluids and tissues is found in several genetic enzyme deficiencies, the homocystinurias. These include cystathionine /3-synlhase deficiency [9], the remethylation defects due to deficiency of MTHF reductase [10], methionine synthase and methionine synthase reductase deficiencies, as well as defects of intracellular cobalamin metabolism [11], namely the cblF, cblC and cblD defects. It is noteworthy that low levels of total Hey (tHcy) have been described in sulphite oxidase deficiency [12]. 

Methionine synthase deficiency (cblC, cblD, cblF, cblE, cblG defects) 100-250... 

Transfer of the methyl group from 5,-adenosylmethi-onine to an acceptor yields S -adenosylhomocysteine (Fig. 18-18, step (2)), which is subsequently broken down to homocysteine and adenosine (step (3)). Methionine is regenerated by transfer of a methyl group to homocysteine in a reaction catalyzed by methionine synthase (step (4)), and methionine is reconverted to 5-adenosyl-methionine to complete an activated-methyl cycle. 

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