THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 268, No. 9, Issue of March 25, pp. 6549-6553, 1993 Printed in U. S. A.

Proofreading and the Evolution of a Methyl Donor Function CYCLIZATION OF METHIONINE TO S-METHYL HOMOCYSTEINE THIOLACTONE BY ESCHERICHIA COLI METHIONYL-tRNA SYNTHETASE*

(Received for publication, October 16, 1992)

Hieronim Jakubowski From the Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103

A cyclic sulfonium compound, S-methyl homocys- teine thiolactone (SMHT), is formed from methionine during in vitro tRNA aminoacylation catalyzed by Escherichia coli methionyl-tRNA synthetase. The mechanism of SMHT formation involves enzymatic de- acylation of Met-tRNA (k = 0.06 s-') and, to a lesser extent, Met-AMP (k = 0.02 s-'). Cyclization of methi- onine, reminiscent of cyclization of homocysteine dur- ing editing, illustrates the limited ability of methionyl- tRNA synthetase to discriminate against the cognate methionine at the editing site designed for the noncog- nate homocysteine. In early stages of biotic evolution, SMHT, a sulfonium compound, may have fulfilled the present day methyl donor function of S-adenosylme- thionine. Existing homologies between methionyl- tRNA synthetase and S-adenosylmethionine synthe- tase indicate evolutionary relatedness of the two pro- teins.

The shape and stability of the genetic code are determined by two pivotal steps of protein synthesis. In the first step, each of the 20 aminoacyl-tRNA synthetases correctly matches a cognate amino acid selected from the 20 protein amino acids (and a few nonprotein ones like homocysteine, homoserine, ornithine, etc.) with its cognate tRNA selected from the 20 tRNA families. In the second step, a correct aminoacyl-tRNA is selected in the codon programmed ribosomal A site. High accuracy of aminoacyl-tRNA synthetases assures proper as- signment of an amino acid to its cognate tRNA and is achieved by a highly specific recognition of tRNA (1) and by proof- reading or editing mechanisms that remove errors in amino acid selection (2, 3).

The editing mechanism of methionyl-tRNA synthetase is directed against homocysteine. Methionine and its immediate metabolic precursor, homocysteine, are present in Escherichia coli at similar concentrations of about 50 p~ (4, 5). As ex- pected from in vitro studies (6, 7 ) , homocysteine is misacti- vated i n vivo at unacceptably high levels (5). Misactivated homocysteine is very efficiently edited both i n vitro ( 7 ) and in vivo (5, 8), which prevents its incorporation into cellular proteins.

A distinctive feature of homocysteine editing is that the enzyme-bound homocysteinyl adenylate (I) is cyclized to yield

*This research was supported by National Institutes of Health Grant GM-27711 and Biomedical Research Support Group Grant 2S07RR05393 and by Foundation of University of Medicine and Dentistry of New Jersey Grant 9-93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

homocysteine thiolactone (11) (Equation 1).

NH.+ +

I I1

Methionyl-tRNA synthetase, as most aminoacyl-tRNA synthetases, has a 2-fold selectivity problem. In the synthetic reaction, it has to discriminate against a noncognate amino acid. In the editing reaction, it has to discriminate against a cognate amino acid. Because discrimination against the non- cognate homocysteine in the synthetic site is limited, discrim- ination against the cognate methionine in the editing site is also expected to be limited. Therefore, a small fraction of methionine would pass through the editing site. This would lead to cyclization of methionine to S-methyl homocysteine thiolactone, analogous to cyclization of homocysteine to hom- ocysteine thiolactone.

Here, it is shown that methionine is cyclized to a sulfonium compound, S-methyl homocysteine thiolactone, by E. coli methionyl-tRNA synthetase. The mechanism of cyclization involves enzymatic deacylation of Met-tRNA and, to a lesser extent, Met-AMP. The data point to a functional and evolu- tionary relationship between metG and metK genes, which is further supported by sequence homologies of the protein products of these genes.

MATERIALS AND METHODS

Methionyl-tRNA Synthetase-Plasmid pGG3 containing the me- thionyl-tRNA synthetase gene subcloned into phagemid vector pTZ18R was obtained from the late L. Schulman (Albert Einstein College of Medicine). The methionyl-tRNA synthetase gene produces a truncated protein that is fully active both in vitro and in vivo (9). Plasmid pGG3 was transformed into E. coli strain rnetG+-l (5) and used as a source of methionyl-tRNA synthetase, purified to homoge- neity by the accepted criteria (7, 9).

Preparation of P'SIMet-tRNA and rH]Met-tRNA-Reaction mix- tures contained a standard buffer (50 mM HEPES, pH 8.0, 5 mM MgClz, 1 mM dithiothreitol, 0.1 mM EDTA), 2 mM ATP, 20 p~ tRNA""' (1460 pmol/AaW; Sigma), 50 p~ [35S]methionine or [methyl- 3H]methionine at 0.1 mCi/ml (1 Ci = 37 GBq; Amersham Corp.), and 50 nM methionyl-tRNA synthetase. After 30 min at 37 "C, the reac- tion was completed at a level of 90% tRNA charged which is the highest possible level (10). The charged tRNA was recovered by repeated precipitations and washed with 70% ethanol until all free radioactive methionine was removed.

Enzymatic Deacylation of Met-tRNA-The reactions were carried out in a standard buffer at 37 "C. In one set of experiments, the

6549

6550 Evolution of a Methyl Donor Function

.

spot 1

Met

Met oxide

Origin

FIG. 1. TLC separation of the products formed from methi- onine in aminoacylation mixtures. Reaction mixtures containing 2.5 p~ Met-tRNA synthetase, 2.5 p~ tRNAwe, 1 mM ATP, and 10 p~ [%]methionine (IO6 Ci/mol) were incubated a t 37 "C for 30 min. Lune 1 , complete reaction mixture; lone 2, -Met-tRNA synthetase; lone 3, -tRNA.

disappearance of radiolabeled Met-tRNA was monitored by trichlo- roacetic acid precipitation. In another set of experiments in which all forms of radioactive methionine were followed, the aliquots were quenched with 1 M formic acid a t 0 "C and analyzed by TLC. TLC Analysis-The TLC system (cellulose plates developed with

butano1:acetic acidwater, 4:1:1, v/v) is the same as the one used before for the determination of aminoacyl adenylates (1 1) and hom- ocysteine thiolactone (7). In experiments with [RsS]methionine or [%S]Met-tRNA, the TLC plates were autoradiographed using XAR- 5 Kodak x-ray film. Quantitation was by liquid scintillation counting. Counting efficiency was 10% for 3H and 60% for 3sS.

RESULTS

A Methwnine-derived Compound Is Formed during Amino- acylation of tRNA'"'--Reaction mixtures containing [3sSs] methionine, ATP, tRNAM", and methionyl-tRNA synthetase were incubated a t 37 "C. Products of the reactions were sep- arated by TLC. An autoradiogram exposed from these chro- matograms is presented in Fig. 1. In addition to ["S]Met- tRNA, which stays at the origin of the TLC plate, one new major 35S compound appeared in the complete reaction mix- ture (Spot I in lane I). Control reaction mixtures without enzyme (lane 2) or without tRNA (lane 3) did not contain this compound. Thus, under these experimental conditions, Spot 1 formed only in the presence of both tRNA and methi- onyl-tRNA synthetase.

Identification of a Methionine-derived Compound as S- Methyl Homocysteine Thiolactone (SMHT)'-Preliminary ex- periments indicated that the new methionine-derived com- pound can be recovered by extraction of aminoacylation mix- tures with toluene. The recovery was essentially quantitative when the mixtures were buffered at pH 8.0. Thus, to establish the identity of Spot 1, aminoacylation mixtures were extracted with toluene and the extracted material was subjected to several chemical and enzymatic tests. The fact that the com- pound of Spot 1 is soluble in toluene at pH 8 indicates that it is more hydrophobic than homocysteine thiolactone (which is essentially not soluble in toluene), a property expected of SMHT.

Treatment of the compound of Spot 1 with NaOH or ammonia (Fig. 2, lanes 2, 3, and 6) but not with 0.1 M HCI (Fig. 2, lane 5) converted it into methionine. Enzymatic hydrolysis with rabbit esterase also converted Spot 1 into methionine (Fig. 2, lane 4 ) . Half-life for spontaneous hydrol-

The abbreviations used are: SMHT, S-methyl homocysteine thio- lactone; AdoMet, S-adenosylmethionine.

P

Met

~ Met oxide

Origin

FIG. 2. TLC resolution of the products of enzymatic and nonenzymatic hydrolysis of Spot I . The complete arninoacylation mixture with [x'S]methionine (see Fig. 1) was extracted with toluene. The solvent was removed by evaporation, and the reRidue was taken up in water and subjected to the indicated treatments at 22 'C for 10 min (reaction volume, 3 pl). Lune I , untreated; lunes 2 and 6, +0.1 M NaOH; lone 3, +1 M ammonia; lane 4, +0.5 unit of rabbit esterane; lone 50.1 M HCI. 75 'C.

1 2 3 4 5

SMHT

SMHTO

Met

Met oxide Origin

FIG. 3. "LC analysis of the oxidation products of SMHT. A preparation of SMHT was treated with 0.1 M HZOz (30 min, 22 'C) and subjected to TLC either directly or after hydrolysis with NaOH. Lune 1 , +H202 followed by NaOH; lane 2, untreated; lone 3. +NaOH; lone 4, +H202; lone 5, +NaOH followed by H,Oz. SMHTO. S-methyl homocysteine thiolactone sulfoxide.

ysis of Spot 1 to methionine at pH 8, 37 "C was 80 min (not shown). In all cases, methionine was the only product of hydrolysis of Spot 1. Based on these properties, the compound of Spot 1 is concluded to be SMHT.

Oxidation of S M H T to the Sulfoxide-A minor spot, mi- grating between SMHT and methionine spots, always accom- panied formation of SMHT. The proportion of this minor compound in the preparations of SMHT gradually increased during storage a t -20 "C. As shown in Fig. 3, a preparation containing 50% SMHT and 50% of a slower migrating com- pound (lane 2) was hydrolyzed by NaOH to equivalent amounts of methionine and methionine sulfoxide (lane 3 ) . Upon oxidation of the original preparation, all SMHT was transformed into a slower migrating compound (lane 4 ) . Both oxidation followed by base hydrolysis (lane 1 ) and base hy- drolysis followed by oxidation (lane 5) gave methionine sulf- oxide. Thus, a slower migrating compound is S-methyl hom- ocysteine thiolactone sulfoxide.

S-Methyl Homocysteine Thiolactone Is a Product of Enzy- matic Deacylatwn of Met-tRNA-As shown in Fig. 4A, enzy- matic deacylation of Met-tRNA led to formation of a major

Evolution of a Methyl Donor Function 6551

A B 1 2 3 4 5 6 7 1 2 3 4 5 6 7

SMHT

SMHTO Met

Met oxide

t Met-tRNA - -

FIG. 4. TLC separation of the products of enzymatic and nonenzymatic deacylation of Met-tRNA. Reactions were carried out at 37 "C in a standard buffer (50 mM HEPES, 5 mM MgCl,, 0.1 mM EDTA, 1 mM dithiothreitol) with 0.25 p~ ["%]Met-tRNA in the presence of 2.5 p~ Met-tRNA synthetase (panelA ) and in its absence (panel R) . Panel A, enzymatic deacylation was carried out for 0,0.25, 1, and 5 min (lanes 1-4, respectively). After 5 min, the products were treated with either 50 mM H202 (lane 5 ) or 0.1 M NaOH (lane 6 ) . Treatments with H202 ( l a n e 5 ) or NaOH (lane 6 ) were terminated before completion. Panel R, nonenzymatic deacylation was carried out for 0, 5, 15, and 45 min (lanes 1-4, respectively). After 45 min, the products were treated with either 50 mM H202 (lane 5) or 0.1 M NaOH (lane 6). Lane 7 in both panels is ["S]S-methyl homocysteine thiolactone sulfoxide ( S M H T O ) standard.

A. : r B. I

- 8 . . "

:3 0 * .,

I= E *

'/I c

5 I < : 2 -

L i

- - . - - n . .

I I

8 l I." q . 2

3" 5 2 . : e

Ttme. '1 * h e . mln

FIG. 5. Time course of SMHT formation in aminoacylation mixtures. Conditions were: pH 8.0, 37 "C, 1 p~ Met-tRNA synthe- tase, 2.4 pM ["]methionine (89,600 (Ci/mol), 1 mM ATP. Panel A, +3 p~ tRNA""; panel B, -tRNA.

product comig-rating with SMHT on TLC plates. This product exhibited all properties of SMHT. Treatments with H,O, or NaOH transformed it to S-methyl homocysteine thiolactone sulfoxide or methionine, respectively (lanes 5 and 6 in Fig. 4A).

Analyses of the products of nonenzymatic deacylation of Met-tRNA were also performed. As shown in Fig. 4R, free methionine was a major product of the nonenzymatic reaction.

Kinetics of S-Methyl Homocysteine Thiolactone Forma- tion-The time course of SMHT formation in the aminoac- ylation mixtures is presented in Fig. 5. The tRNA-dependent reaction reached a plateau when all methionine was trans- formed into SMHT (Fig. 5 A ) . At 2.4 PM methionine, the reaction in the presence of tRNA (Fig. 5 A ) was 200-fold faster than the reaction in its absence (Fig. 5B) . Increasing methi- onine concentration to 50 PM led to a 10-fold increase in the rate of SMHT formation in the absence of tRNA, whereas the rate of the tRNA-dependent reaction was not significantly affected. A further increase in methionine concentration did not accelerate the reaction. However, it was noticed that inclusion of inorganic pyrophosphatase in reaction mixtures accelerated the rate of SMHT formation in the absence of tRNA (but not in the presence of tRNA) even a t a saturating concentration of methionine. Thus, to obtain rate constants for tRNA-independent SMHT formation, the measurements were carried out a t 50 p~ methionine in the presence of 5

units/ml yeast inorganic pyrophosphatase (Sigma). Under these conditions, the rate constant for the tRNA-independent reaction was 0.02 s".

The time course of SMHT formation during deacylation of Met-tRNA by methionyl-tRNA synthetase is presented in Fig. 6. The rate of SMHT formation was equal to the rate of deacylation of Met-tRNA. The rate constant for enzymatic deacylation measured with an excess enzyme was 0.058 s-l (Table I). Small amounts of methionine were formed as a result of spontaneous deacylation of Met-tRNA ( k = O.OOO9 S").

Inhibition of Enzymatic Deacylation of Met-tRNA by Amino Acids-Previous results indicate that editing of homocysteine by methionyl-tRNA synthetase does not depend on tRNA and may not involve an editing site separate from a synthetic site ( 7 ) . In contrast, valyl- (12), isoleucyl- (13), and phenyla- lanyl-tRNA synthetases (14) were proposed to possess a sep- arate deacylating site for editing. The conclusion about the separate deacylating site was based on the lack of significant inhibition, by an amino acid or aminoacyl adenylate, of en- zymatic deacylation of aminoacyl-tRNA.

As shown in Table I , all amino acid substrates, both cognate and noncognate, of methionyl-tRNA synthetase inhibited enzymatic deacylation of Met-tRNA a t 2S "C. The obsewa- tion that homocysteine alone inhibits enzymatic deacylation of Met-tRNA at 25 "C but not at 37 "C may indicate that its affinity for the enzyme is greater at 2.5 "C than at 37 "C. At 37 "C, the inhibition was more pronounced in the presence of aminoacyl adenylates formed in situ. Thus, a single active site is involved in both activation and editing of methionine as well as noncognate amino acids bv methionyl-tRNA synthe- tase.

Homologies in Primary Structure between Methionyl-tRNA Synthetase and AdoMet Synthetase-Methionyl-tRNA syn- thetase and AdoMet synthetase share two common substrates.

- - /- - _ I

? d . . . ~ - _ _ . - . - -me m7-

FIG. 6. Time course of SMHT formation durinK enzymatic deacylation of Met-tRNA. Conditions were: pH 8.0. 37 "C, 1 p~ Met-tRNA synthetase, 2.7 p~ ['HJMet-tRNA (2000 Ci/rnol). 0. ['HI SMHT; 0. 13H]Met-tRNA; x, ["]Met.

TABLE I Inhibition of enzymatic deacylation of Met-tRNA by amino acidp

Conditions were: pH 8.0, 1 p~ ['HJMet-tRNA"" (2000 Ci/mol), 2.5 pM Met-tRNA synthetase. 5 mM amino acid and 1 mM ATP were present where indicated. Rate conRtants as ahown were corrected for nonenzymatic deacylation of Met-tRNA ( k = O.OOO9 R-' at 37 'C and 4 0 " a" at 25 "C).

k

37 'C 25 'C Additionn

In3 X # - I

None 58 9.6 Methionine 1.9 4 . 1 Methionine, ATP 0.6 <0. 1 Homocysteine 58 2.9 Homocysteine, ATP 19 Norleucine 3.3 Ethionine 1 .o

6552 Evolution of a Methyl Donor Function

methionine and ATP. Both enzymes catalyze formation of a sulfonium compound. The present day methyl donor, AdoMet (22), and a putative methyl donor, SMHT (this work), are made in reactions catalyzed by AdoMet synthetase and me- thionyl-tRNA synthetase, respectively. Because of these func- tional similarities, amino acid sequences of the two proteins were compared. As shown in Fig. 7, two regions of methionyl- tRNA synthetase in the middle portion of the protein (amino acids 192-249 and 322-455) show significant homologies with the middle portions of AdoMet synthetase (amino acids 86- 143 and 154-283).

The methionyl-tRNA synthetase sequence segment span- ning amino acids 322-455 is the COOH-terminal region char- acterized by the KMSKS sequence. This segment is one of the two segments of high homology between class I aminoacyl- tRNA synthetases (23, 24). The segment of methionyl-tRNA synthetase spanning amino acids 192-249 contains portions of the ATP/Met binding domain (9, 25), and the segment 322-455 contains portions of both the ATP/Met binding domain (amino acids 322-359) (9,25-27) as well as the tRNA binding domain (amino acids 361-455) (25). Two ultimate amino acids, Lys-335 and Ser-336, in the KMSKS sequence of methionyl-tRNA synthetase correspond to Lys-167 and Ser-168, respectively, of AdoMet synthetase. The lysine resi- due 335 of methionyl-tRNA synthetase is implicated in inter- actions with the pyrophosphate moiety of ATP (27), a func- tion that is likely to be conserved in AdoMet synthetase. The aligned segments of methionyl-tRNA synthetase and AdoMet synthetase share 21-22% of identical amino acids. This degree of similarity in region I is at the upper end of the similarities among corresponding segments of class I aminoacyl-tRNA synthetases (7-29%, Ref. 24). No significant homologies were found between other class I aminoacyl-tRNA synthetases and AdoMet synthetase.

DISCUSSION

This work establishes that cyclization is a specific editing mechanism by which methionyl-tRNA synthetase removes errors in amino acid selection. I t also demonstrates functional and evolutionary relationships between methionyl-tRNA syn-

Reglon I

M C t G : 322

MetK: i54

MetG 371

MetK 199

M e t G 420

MetK 2 4 6

VHGYVTVNGAKMSKSRGTFIKASTWLNHFDADSLKYYYtaklSSRIDDI 3 7 0

VRKNGTLRVRPDAKSQ'JTFFYDDGKIVGIDAWLSTQII . . . . SEEIDGK 198

DLNLEdfVQRVNADIVNKWNLASRNAGFINKRFDGVLASELADPQLYK 419

SLQEA..VMEEIIKPILPAEWLTSATKFFINPTGRFVIGGPMGDCGLTG 2 4 5

TFTDAAEVIGEAWESREF..GICAVREIMALADLANRW 455

RKIIVDTTGGMARHGGGAfsGKDPSKVDRSAAYAARYV 283

Reglon I1

M e t G : 192 FDLPSFSEMLQAWTRSGALQEGVRNKMQEWFESGLQG~ISRDAPYFGFEIPNAP 249

MetK: 8 6 FDRNSCAVLSAIGKQSPDINQGVDRADPLEQGAGUQGLDVSATQLMKPTCLMPAP 1 4 3

FIG. 7. Amino acid sequence homologies between E. coli methionyl-tRNA synthetase (MetG) (29) and AdoMet synthe- tase (MetK) (30). The sequences were aligned with the Altschul program (31). Lower case letters indicate unaligned amino acids, and dots indicate gaps. The alignment shown here has been obtained with a unit odds scoring matrix. Monte Carlo simulations yielded scores from 5.0 to 6.1 and from 3.3 to 5.5 for similarity Regions I and I I , respectively, with three different scoring matrices. A score of >3 indicates significant similarity. For comparison, the Monte Carlo score for the corresponding alignment of Region I of methionyl-tRNA synthetase (29) and the most closely related cysteinyl-tRNA synthe- tase (23) was 6.6-7.3.

thetase and AdoMet synthetase genes. During editing, methionine is cyclized to SMHT, similar to

the cyclization of homocysteine to homocysteine thiolactone (Equation 1) described before (5, 7). The major route of SMHT (IV) formation involves enzymatic deacylation of Met-tRNA (111) by methionyl-tRNA synthetase (Equation 2). The enzyme-bound Met-AMP yields SMHT at a slower rate than the enzyme-bound Met-tRNA.

NH: I

NH~+ I - h = o + tRNA

L S L S ' + \

H3 (Es. 2)

I11 IV

The similar natures of cyclization reactions with homocys- teine and methionine indicate that both occur on the same editing site of the enzyme. With the cognate substrate methi- onine, editing activity is mostly directed toward the enzyme- bound Met-tRNA, the enzyme-bound Met-AMP being 3-fold more stable. With the noncognate homocysteine, editing is directed toward the enzyme-bound Hcy-AMP (7), and there is no evidence for transient formation of Hcy-tRNA during editing (6). It is not quite clear how the 3' terminus of Met- tRNA can be accommodated at the editing site designed for Hcy-AMP and how both can yield mechanistically similar products. Perhaps cyclization of Hcy-AMP involves transient transacylation from the 5"phosphate to 3'-OH of the aden- ylate.

Support for the physical relatedness of the editing and synthetic sites of methionyl-tRNA synthetase comes from the observations that its amino acid substrates, the cognate me- thionine and three noncognate amino acids (homocysteine, norleucine, and ethionine), inhibit enzymatic deacylation of Met-tRNA.

Cyclization during editing may be a general editing mech- anism used also by other aminoacyl-tRNA synthetases. In fact, homocysteine is cyclized to the thiolactone during editing by isoleucyl-, valyl- (7), and leucyl-tRNA synthetases (15). Homoserine is cyclized to homoserine lactone during editing by valyl- and isoleucyl-tRNA synthetases (3). Hydroxyana- logues of valine, leucine, and isoleucine are cyclized to corre- sponding lactones by leucyl- (15) and valyl-tRNA synthetases (16). Although enzymatic deacylation has been described for several aminoacyl-tRNAs (14,17-21), only in two more cases, deacylation of Ile-tRNAPhe (14) and Tyr-tRNAPhe (17) by phenylalanyl-tRNA synthetase, were the products carefully analyzed and determined to be intact amino acids. Reexami- nation of the products of reactions of the synthetases with amino acids, .including enzymatic deacylation reactions, is certainly needed to establish how general this editing mech- anism is.

The synthetic and editing reactions of methionyl-tRNA synthetase ( E ) with methionine ( M e t ) and homocysteine (Hcy) are depicted in Scheme 1 ( K , and kcat values are from Ref. 7 and this work). The accuracy of Met-tRNA synthesis is determined at two points in the pathway. In the synthetic site of methionyl-tRNA synthetase, methionine is selected (bound) preferentially over homocysteine. The discrimination factor of 200 against homocysteine is caused by differences in binding energies. E . Met and E . Hcy have similar reactivities

Evolution of a Methyl Donor Function 6553

SMHT quence homologies between methionyl-tRNA synthetase and AdoMet synthetase proteins (Fig. 7) support this scenario. The segments of methionyl-tRNA synthetase homologous to

30 pM 93 5" 3 s" AdoMet synthetase span portions of both the ATP/Met and E + Met E . M e t " E . M e t - A M P " E . M e t - t R N A tRNA binding domains (25). This is consistent with the

observation that a putative ancestral methyl donor, SMHT, 5 mM 81 5" ? is formed mostly from Met-tRNA and may indicate that a

E + HCY E.HcY ;E.HCY-AMP=E.H~Y-~RNA portion of the tRNA binding domain of an ancestral methio- nyl-tRNA synthetase became the ATP binding domain of the present day AdoMet synthetase. The lysine residue 335 of methionyl-tRNA synthetase, which is known to interact with

Hcy thiolactone the pyrophosphate moiety of ATP and the 3'-end of tRNA

2.4 5-l \ / SCHEME 1 (27), is conserved in AdoMet synthetase.

REFERENCES

2. Fersht, A. R. (1986) in Accur zn Molecular Processes: Its Control and Relevance to Living System Zrkwood, T. B. L., Rosenberger, R. F., and Galas, D. J. e&) p 67 82 Chapman and Hall, London

in formation of a respective enzyme-bound aminoacyl aden- 1. Schulman, L. H. (1991) Pmg. Nuc@ic Acid Res. Mol. BWL 4 1 , 2 3 4 7 ylate. E. Met-AMP is stable and proceeds to E . Met-tRNA.

thiolactone (7), and no Hcy-tRNA is formed (6). Destruction 4. Dev, I. K. & Harvey, R. J. ( 1 9 ~ ) J . ~ i o l . them. 259,8402-~406

removal of an error. A small fraction of Met-tRNA (and an 7. Jakubowski, H. & Fersht, A. R. (1981) Nucleic Acids Res. 9,3105-3117 even smaller fraction of Met-AMP) is also destroyed with the :: $ ~ ~ ~ 6 ~ p 2 ~ ! g ~ ~ & ~ , ~ & ' ~ ' ~ g 2 5 ~ ~ n i e , s, (1991) Biochemistry formation of SMHT. This is also necessary but undesirable 30,9569-9575

for a fraction of Met-tRNA destroyed because of editing is 29-37 0.02 (calculated from the rate constants for synthesis and 13. Schreikr, A. A. & ScEmAel, P. R. (1972) Biochemistry 11,1582-1589

12. Fersht A. R. & Din all C. (1979) Biochemistry 18,1238-1245

fraction of Met-tRNA deacylated in vivo is only 3 X (28). 15. Englisch, S., Englisch, U., von der Haar, F. & Cramer, F. (1986) Nucleic

The &scovery of SMHT synthesis by methionyl-tRNA 16. Englisch-Peters, s., von der Haar, F. & Cramer, F. (1990) Biochemistry 2 9 , Acids Res. 14 , 7529-7539

synthetase is also relevant to the evolution of a methyl donor 17. Lin, S. X., Baltzinger, M. & Remy, P. (1984) Biochemistry 23,4109-4116 function in biological systems. SMHT is a sulfonium corn- i:: FLzf; f : ~ ( S ~ ~ ~ ~ ~ ~ ~ Z f ; ( ~ ~ ~ 2 1 ) ~ 5 ~ ~ ~ O C h e m . 247* 2961"2963 pound and is structurally related to another sulfonium com- 20. Fersbt A. R. & Kaethner M. (1976) Biochemistry 16,3342-3346 pound the present day methyl donor AdoMet (22). In early 22. Cantoni, G. L. (1953) J. io^ C h m . 204,403416

21. von dkr Haar, F. & Cram&, F. (1976) Biochemistry 16 , 4131-4138

thetase may have provided activated methionine both for 24. Nagel, G. M. & Ddi t t l e , R. F. (1991) pm. Natl. ad. sei, u, s. A, 88,

SMHT). Because an efficient and accurate protein synthesis 26. Ghosh,'G., Brunie, S. & Schulman, L. H. (1991) J. Bid. Chem. 266,17136-

a separate system for providing methyl donor function had to M O L SA. 2'17,465-46 evolve. Thus, the Present day AdoMet synthetase has arisen, 29. Dardel, F., Pa at, G. Blan uet, s. ( 1 9 ~ ) J. Bacterial. 160,1115-1122

ancestral methionyl-tRNA synthetase gene. Existing se- 31. Altschul, F. S. & Erickson, B. w. (1986) Math. ~ i o l . 48,603-616

In E'Hcy-AMP is destroyed to give homocysteine 3, Jakuhwski, H. & GOY&&, i, (1992) Mkmbkl. Rev, 56 ,412429

of Hcy-AMP is necessary and desirable because it results in 5. Jakubowski, H. (1990) Proc. Natl. ACad. sci. u. s. A. 87,4504-4508 6. Fersht, A. R. & Dingwall, C. (1979) Biochemistry 18, 1250-1256

and is, therefore, expected to be minimized* An upper limit 11. Jakubowski, H., Pastuszyn, A. & Loftfield, R. B. (1977) AmL Biochem. 82, 10. Jakubowski, H. (1978) Biochim. Biophys. Acta 618,345-350

&acylation of Met-tRNA). As has been shown elsewhere, the 14. Yams, M. (1972) Proc. A d . Sei. U. S. A. 69,1915-1919

7953-7958

stages of biotic evolution, an ancestral methionyl-tRNA syn- 23. Hou,,Y.-M., Shiba, K., Mottes, C. & Schimmel, P. (1991) Proc. Natl. Acad. SCL. U. S. A. 88 976-980

synthesis (as Met-tRNA) and for (as 25. Brunie S., Zelwer, C. & Rider, J.-L. (1990) J. Mol. Bwl. 216,411-424 8121-8125

requires that SMHT synthesis from Met-tRNA be minimized, 27. Mechulam Y. Dardel, F. Le Corre, D., Blanquet, S. & Fayat, G. (1991) J . 17141

28. Jakubowski H. (1993 FASEB J. 7 , 1%172

possibly by duplication and subsequent mutations of the 30. Markham, G.L., DeParasis,% & Gatmaitan, J. (1984) J. Biol. Chem. 269 , 14505-14507