Aminoacyl-tRNA Synthetases

Louis Pasteur, Strasbourg, France (Present address: Albert Einstein College John G Arnez, Universite of Medicine, Bronx, New York, USA) Louis Pasteur, Strasbourg, France Dino Moras, Universite

Introductory article
Article Contents
. Introduction . Class I and Class II of Aminoacyl-tRNA Synthetases . Catalytic and Special RNA Recognition Domains . tRNA-binding Modes of the Two Classes

Aminoacyl-tRNA synthetases catalyse a key reaction in protein biosynthesis. They match the 20 amino acids to the genetic code by specifically attaching them to their adaptors, transfer ribonucleic acid (tRNA) molecules.

Introduction
Aminoacyl-tRNA synthetases catalyse the highly specic attachment of amino acids to their corresponding adaptor molecules, the transfer ribonucleic acids (tRNA). The attachment reaction is called aminoacylation and proceeds in two steps (reactions [I] and [II]), in the presence of divalent magnesium (Mg 2+) ions. amino acid 1 ATP 0 aminoacyl-adenylate 1 PPi [I] aminoacyl-adenylate 1 tRNA 0 aminoacyltRNA 1 AMP ATP is adenosine triphosphate, AMP is adenosine monophosphate and PPi is inorganic pyrophosphate (diphosphate). In step [I], the amino acid is activated through the formation of aminoacyl-adenylate, which is a mixed anhydride of the a-carboxylate group of the amino acid and the a-phosphate group of ATP. The pyrophosphate is released, while the aminoacyl-adenylate remains bound to the active site of the enzyme. In step [II], the amino acid portion is transferred to the 2 or 3 sugar hydroxyl group of the 3-terminal adenosine nucleotide of the tRNA. This is the rate-limiting step. Aminoacylation consumes energy, which is supplied by the hydrolysis of ATP. Both steps are bimolecular nucleophilic substitutions, which means that two molecules react, of which one is the attacking nucleophile that substitutes another group already present; the latter becomes the leaving group. In order for the aminoacyl-tRNA synthetases to catalyse the reaction, they must position the substrates in correct mutual orientations and release the reaction products in a timely fashion. They must also stabilize the reactants and transition states of both steps. First, the amino acid and ATP are bound. Both of these substrates possess negatively charged groups that are to be brought close together and react: the a-phosphate of ATP and the a-carboxylate group of the amino acid. The charge of the phosphate group has to be neutralized, since the amino acid carboxylate is the attacking nucleophile. The [II]

charge neutralization is accomplished by positively charged protein side-chains and divalent metal cations; these withdraw electrons from the phosphate group such that it is receptive to the pair of electrons of the nucleophile. The resulting transition state is pentacoordinate at the aphosphorus, which means that there are ve chemical groupings transiently attached to the phosphorus atom: the amino acid, adenosine, pyrophosphate and two oxygens. It also has many negative charges that are similarly stabilized by the same groups of the enzyme: two on the a-phosphate, one on the b-phosphate and two on the g-phosphate. It is resolved through the dissociation of the pyrophosphate. The aminoacyl-adenylate remains in the active site of the enzyme and needs to be protected from reacting with water. This is an SN2 reaction (substitution, nucleophilic, bimolecular) and results in an inversion of conguration at the a-phosphorus. Its mechanism corresponds to an in-line associative nucleophilic substitution; it is associative as opposed to dissociative because the leaving group is displaced after the nucleophilic attack and transition state formation rather than before, and in-line because the nucleophile attacks on the side directly opposite the leaving group. Second, the tRNA is bound such that its terminal ribose (sugar) hydroxyls are close to the carbonyl carbon of the aminoacyl-adenylate. Some of the tRNA phosphate charges need to be balanced in order for it to bind. The negatively charged phosphate of the aminoacyl-adenylate is neutralized as well. However, this step does not involve phosphates but a hydroxyl and a carbonyl group. This too is a nucleophilic substitution and takes place at the acarbonyl carbon of the amino acid moiety of the mixed anhydride. In this case, the hydroxyl is the nucleophile; the carbonyl carbon has a slight positive formal charge and is thus a good electrophile. The transition state consists of four groups covalently bound to the a-carbonyl carbon: the amino acid, the tRNA, the AMP and the hydroxyl derived from the carbonyl group. The AMP is the leaving group in this substitution. The resolution of the transition state results in an ester linkage between the ribose hydroxyl
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Aminoacyl-tRNA Synthetases

and amino acid carboxyl groups. Finally, the aminoacylated tRNA is released. Since there are 20 amino acids specied by the genetic code, there are generally 20 aminoacyl-tRNA synthetases in a given cell. Each enzyme is highly specic for its amino acid and matching set of tRNAs called isoacceptor tRNAs; these three components constitute a cognate aminoacylation system. All of these enzymes catalyse the same basic reaction and share a common substrate, ATP. Amino acids have the same core chemical groups and tRNAs are folded in the same L-shaped three-dimensional structures. These common features enable these compounds to engage as substrates in other reactions of protein biosynthesis. However, amino acids have dierent side-chains, each of which is recognized by its proper (cognate) aminoacyltRNA synthetase and rejected by the remaining 19. Each tRNA harbours within the common structural framework a variety of features that form its separate identity; some of them enable its cognate enzyme to accept it (positive determinants) while others cause all other aminoacyltRNA synthetases to reject it (negative determinants). Often an amino acid is attached to several tRNA species, since it is specied by several three-letter keywords called codons in the genetic code. All these tRNA species form a family of isoacceptors that share the same identity and are recognized by the same aminoacyl-tRNA synthetase. Most identity nucleotides or elements are found in the acceptor arm or the anticodon arm of the tRNA; occasionally they can also be found in the structural core region formed by the D and T arms. Some enzymes are more specic for a particular structural feature of a given tRNA than the nucleotide sequence, i.e. the exact nature of the nucleotide bases. Identity elements are also distributed dierently in dierent isoacceptor tRNA families, which adds an additional aspect to the specicity of recognition and thus further strengthens it.

Table 1 Classication of aminoacyl-tRNA synthetase Quaternary structure OH aminoacylated Class I Active site: nucleotide-binding fold Motifs: HIGH, MSK LeuRS IleRS ValRS CysRS MetRS GluRS GlnRS ArgRS TyrRS TrpRs Class II Active site: antiparallel b sheet Motifs: 1, 2, 3 HisRS ProRs SerRs ThrRS GlyRS AspRS AsnRs LysRS AlaRS PheRS Ribose

a a a a a2 a a a a2 a2

2 2 2 2 or 3 2 2 2 2 2 or 3 2

a2 a2 a2 a2 a2 a2 a2 a2 a4 (ab)2

3 3 3 3 3 3 3 3 3 2

Class I and Class II of Aminoacyl-tRNA Synthetases

As individual members of these enzymes were discovered and examined, they were noted for their diversity in amino acid sequence, size and quaternary structural organization, ranging from small monomers (single protein subunits) to large tetramers (four protein subunits). This seemed paradoxical in view of the same basic reaction that they catalyse and similar substrates they employ. Some common features were nevertheless discovered, arising from two sources of information. First was the expanding database of amino acid sequences for all aminoacyl-tRNA synthetases obtained from several species. Second was the determination of the three-dimensional structures of a few key enzymes of this family. The common features, however, did not extend over the entire
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family. Some applied to one half and some to the other half, partitioning the family into two classes, whose denition is based on two dierent sets of limited amino acid sequence similarities and two active site architectures (Table 1). The two classes represent two solutions to the same problem of aminoacylation (Figure 1).

Class I
Class I aminoacyl-tRNA synthetases are primarily monomeric proteins and share conserved amino acid sequence motifs histidine-isoleucine-glycine-histidine (abbreviated His-Ile-Gly-His or HIGH) and lysine-methionine-serinelysine-serine (Lys-Met-Ser-Lys-Ser or KMSKS). The active-site architectures are based on the classical nucleotide-binding fold, a parallel b sheet surrounded by a helices, also known as the Rossmann fold, which they share with many other nucleotide-binding proteins. This structure constitutes a platform for binding ATP. The two

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Aminoacyl-tRNA Synthetases

Figure 1 Active-site domains of aminoacyl-tRNA synthetases complexed with the acceptor arms of their cognate tRNAs. Shown also are ATP and key interactions with tRNA. (a) Class I glutaminyl-tRNA synthetase. The nucleotide-binding fold (the Rossmann fold) is shown in yellow and the acceptor binding insertion is light blue. The two class I characteristic motifs HIGH and KMSKS are highlighted in red and blue, respectively. (b) Class II aspartyl-tRNA synthetase. The three class II characteristic motifs are highlighted: motif 1 in red, motif 2 in green and motif 3 in blue. The insertion is shown in light blue.Adapted from Arnez JG and Moras D (1997) Trends in Biochemical Sciences 22: 211 216.

conserved motifs are part of the fold and interact with the nucleotide (Figure 1a). These enzymes bind their cognate amino acids in open and relaxed binding pockets. They bind ATP in an extended conformation, which is similar to other proteins that have the Rossmann fold. The adenine of ATP lies against the glycine of the HIGH motif. The histidines of the HIGH motif and the lysines of the KMSKS motif interact with the triphosphate of ATP. The triphosphate coordinates at least one divalent metal cation. These residues and the cation withdraw electrons from the a-phosphate so that the amino acid a-carboxylate can attack nucleophilically at the phosphorus. These groups also stabilize the transition state of the rst reaction (step [I]). The aminoacyladenylate has a bent conformation, which can be explained by the amino acid approaching from the side in order to react with the a-phosphate of ATP. In the second reaction (step [II]) the amino acid is transferred to the 2 hydroxyl of the terminal ribose of tRNA, for this hydroxyl is the closest to the aminoacyl-adenylate. The three-dimensional structures of seven class I aminoacyl-tRNA synthetases are known; these are the enzymes for tyrosine, methionine, glutamine, tryptophan, glutamate, arginine and isoleucine.

Class II
Class II aminoacyl-tRNA synthetases are primarily dimeric proteins and share three amino acid sequence motifs, labelled motif 1, 2 and 3. The active-site architectures are based on a newly discovered fold observed to date only in these enzymes and a few related proteins. The fold is a seven-stranded antiparallel b sheet surrounded by a helices and represents a new platform for binding ATP. Motif 1 consists of an a helix followed by a b strand and contains a conserved proline; it forms a major part of the dimer interface. Motif 2 consists of two b strands connected by a long loop, and motif 3 comprises a b strand followed by an a helix; the two motifs contain a number of conserved amino acid residues involved in amino acid and ATP positioning and catalysis (Figure 1b). Class II enzymes bind their cognate amino acids in specic rigid binding pockets. Unlike class I enzymes, they bind ATP in a unique bent conformation. The adenine of ATP lies against the conserved phenylalanine of motif 2. The conserved arginines of motif 2 and 3 interact with the triphosphate of ATP, the rst motif 2 arginine with the aphosphate, and the motif 2 loop and motif 3 arginines with the g-phosphate. The enzymeATP complex generally coordinates three metal ions, two between the b- and gphosphates and one between the a- and b-phosphates. The rst motif 2 arginine and the metal ion coordinated to the a-phosphate withdraw electrons from the a-phosphate and thus make it electrophilic (seeking electrons). The nucleo3

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Aminoacyl-tRNA Synthetases

philic carboxylate of the amino acid can thus attack at the a-phosphorus. The motif 2 and 3 arginines and the metal ions also stabilize the transition state of the rst reaction (step [I]). The aminoacyl-adenylate has an extended conformation, which can be explained by the amino acid approaching head-on in order to react with the aphosphate of ATP, since the b- and g-phosphates are bent away. In the second reaction (step [II]) the amino acid is transferred to the 3 hydroxyl of the terminal ribose of tRNA, for this hydroxyl is the closest to the aminoacyladenylate. The three-dimensional structures of eight class II aminoacyl-tRNA synthetases are known; these are the enzymes for serine, aspartate, lysine, phenylalanine, histidine, glycine, proline and asparagine.

Catalytic and Special RNA Recognition Domains

Aminoacyl-tRNA synthetases are modular enzymes (Figure 2). The core domain or module contains the active site and denes the class according to the architecture of the site. Additional modules are attached to the core; many of these interact with various parts of tRNA molecules. Among these are domains that interact with the anticodon loop of tRNA, insertions that form additional contacts with the acceptor arm of tRNA, and modules that recognize certain specic features of a given tRNA. These extra modules diversify the two basic architectures and ensure specic tRNA recognition. Furthermore, some enzymes possess modules that carry out editing functions

Figure 2 Modular organization of aminoacyl-tRNA synthetases and mirror symmetrical binding modes characteristic of the two classes. The tRNAs of glutaminyl-tRNA synthetase:tRNAGln complex (class I, left) and aspartyl-tRNA synthetase:tRNAAsp complex (class II, right) were superposed on tRNAPhe (the first tRNA known in three-dimensional structural detail and commonly used as reference, middle). The two enzymes were then translated away from tRNAPhe for this picture; the variable loop and CCA end are highlighted in blue and green, respectively. The enzyme modules are rendered as follows: the core active-site domains in yellow, the active-site insertions in light blue, the linkers in green and the anticodon-binding domains in orange. Adapted from Arnez JG and Moras D (1997) Trends in Biochemical Sciences 22: 211 216.

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or have roles not directly relevant to aminoacylation. Additional similarities in the core domains that extend beyond the denition of classes and the nature of the added modules further partition each class into three subgroups. These subgroups are also loosely associated with the type of amino acid that is attached to the tRNA.

Class I
One subgroup of class I aminoacyl-tRNA synthetases comprises the two enzymes for aromatic amino acids tyrosine and tryptophan. The two enzymes are dimeric and cannot function as monomers; only one of the two active sites is functional at any one time. These enzymes have insertions in the core nucleotide-binding fold but no additional tRNA-binding module. The second monomer of the dimer functions as the anticodon-binding domain while the rst monomer performs the catalysis. The second subgroup could conceivably contain the three enzymes specic for charged amino acids arginine and glutamate, and polar amino acid glutamine; they can activate the amino acids into their respective adenylates only if they have bound tRNA rst. The enzymes of the third subgroup would then comprise the enzymes that are specic for mostly aliphatic or sulfur-containing amino acids (isoleucine, leucine, valine, cysteine and methionine). Enzymes of both second and third subgroups have insertions in the nucleotide-binding domain for specic contacts with the acceptor arm of tRNA and dedicated anticodon-binding modules. The latter two subgroups are nevertheless not very well dened, which means their membership may change as more structural information becomes available.

The acceptor arm of tRNA is recognized by the catalytic module, which consists of the central nucleotide-binding fold into which is inserted a special accessory subdomain. Specic elements of this module interact with the minor groove of the end of the acceptor arm double helix and the cytidine-cytidine-adenosine (CCA) of the 3 terminus of the tRNA (Figures 1a and 3a). The CCA end is bent into the active site, essentially forming a hairpin that is stabilized by an intramolecular hydrogen bond. Only in this way can the 2 hydroxyl group of the terminal adenosine come within range to accept the amino acid from the aminoacyladenylate. With the possible exception of tyrosyl-tRNA and tryptophanyl-tRNA synthetases, class I enzymes possess anticodon-binding domains, which are attached at the Cterminal end of the core module. The anticodon-binding domain of glutaminyl-tRNA synthetase is a double b barrel (Figure 2, left), whereas those of glutamyl-tRNA, methionyl-tRNA and isoleucyl-tRNA synthetases are ahelical bundles. When glutamine-specic tRNA (tRNAGln) binds to glutaminyl-tRNA synthetase, its anticodon undergoes a substantial conformational change. The nucleotide bases spread such that they maximize contacts with the enzyme and thus enable the enzyme to recognize the specic nature of the bases (Figure 3a). These contacts include specic hydrogen bonds and van der Waals interactions. Analogous conformational changes are likely to occur in other enzymetRNA complexes, although their three-dimensional structures are not presently known. Isoleucyl-tRNA synthetase needs to specically recognize isoleucine and discriminate against valine. The two amino acids are very similar and are both activated in the

Figure 3 Areas of tRNAs that interact with cognate aminoacyl-tRNA synthetases. In each case, nucleotides whose bases form direct hydrogen bonds with the enzyme are shown in red, nucleotides that enable the tRNA to attain the conformation needed for productive interaction with the enzyme are drawn in green, and the portions of the phosphodiester backbone in contact with the enzyme are highlighted as purple spheres. (a) tRNAGln, (b) tRNAAsp and (c) tRNASer.

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rst reaction into their corresponding aminoacyl-adenylates. A second domain, which is inserted into the core catalytic domain, has a binding site that is specic for the noncognate valyl-adenylate; the amino acid recognition portion of the site is too small to accommodate isoleucine, which is larger than valine by a methyl group. Valyladenylate is then hydrolysed, which prevents valine from getting attached to isoleucine-specic tRNA (tRNAIle). Related enzyme valyl-tRNA synthetase also possesses an editing function. It has to distinguish between valine and threonine. These amino acids have approximately the same size; however, threonine has a hydroxyl where valine has a methyl group. The editing site must therefore specically bind this hydroxyl and thus exclude valine. As a result, only valine is attached to valine-specic tRNA (tRNAVal).

Class II
One subgroup of class II aminoacyl-tRNA synthetases is specic for the charged amino acids aspartate and lysine, and polar amino acid asparagine; these enzymes have similar anticodon-binding modules attached to the Ntermini of their active site domains. The second subgroup charges small and polar amino acids (serine, histidine, proline, glycine and threonine); these enzymes, with the exception of seryl-tRNA synthetase, have similar anticodon-binding domains attached to the C-termini of their core modules. The third subgroup comprises multidimeric enzymes, which are specic for alanine and phenylalanine; some prokaryotic glycyl-tRNA synthetases are also multidimeric and may belong to this subgroup. Subgrouping in this class is better dened than that in class I and is clearly related to additional architectural features of these enzymes. The acceptor-binding function of class II enzymes is part of the class II core domain architecture. The motif 2 loop interacts with the major groove of the end of the acceptor arm double helix (Figures 1b and 3b). The CCA end inserts straight into the active site and is held in place by structural elements surrounding the active site. The disposition of the aminoacyl-adenylate, which is roughly parallel to that of the CCA end, obviates the need for the CCA end to bend. The 3 hydroxyl of the terminal adenosine comes within the necessary range for accepting the amino acid from the aminoacyl-adenylate. Two well-dened anticodon-binding domains have been observed among this class of enzymes. Aspartyl-tRNA, asparaginyl-tRNA and lysyl-tRNA synthetases share a ve-stranded b barrel anticodon-binding domain that is attached to the N-terminal end of the core module (Figure 2, right). The fold of this domain is similar to that of some other nucleic acid-binding and even oligosaccharidebinding proteins, for example, major cold shock protein, ribosomal protein S17, staphylococcal nuclease and some enterotoxins. Histidyl-tRNA, glycyl-tRNA, prolyl-tRNA
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and threonyl-tRNA synthetases have a mixed a helix b strand structure with a central ve-stranded mixed b sheet surrounded by three a helices. The domain is attached to the C-terminal end of the catalytic module. When aspartic acid-specic tRNA (tRNAAsp) binds to aspartyl-tRNA synthetase, its anticodon undergoes a dramatic conformational change so as to maximize contacts with the anticodon-binding domain and thus enable the domain to recognize the specic nature of the nucleotide bases. These contacts include specic hydrogen bonds and van der Waals interactions. Analogous conformational changes are likely to occur in other enzymetRNA complexes, to varying degrees. Seryl-tRNA synthetase does not interact with the anticodon of its cognate tRNAs (Figure 3c). Its family of isoacceptor tRNAs possesses a variety of anticodon sequences. Instead, it has a special coiled-coil module attached at its N-terminal end that recognizes a specic feature of serine-specic tRNA (tRNASer), namely the large extra arm in the place of the variable loop. This arm is an important structural element, although the enzyme makes only one nucleotide base-specic contact with the helical portion of the arm. In addition, the enzyme recognizes the acceptor arm and part of the T and D arms. Most of these enzymes have additional insertion domains that may interact with the tRNA. Glycyl-tRNA synthetase has an insertion between motifs 1 and 2, while aspartyl-tRNA, lysyl-tRNA and histidyl-tRNA synthetases have insertions between motifs 2 and 3. The structures of the latter three are a helical. These domains may also vary from one division of the living world to another, as has been observed in the case of aspartyl-tRNA synthetases, where enzymes from prokaryotic organisms have large insertions and those from eukaryotic and archaeal organisms have small insertions. Phenylalanyl-tRNA synthetase is an (ab)2 tetramer, i.e. a homodimer of two a b dimers. The smaller a subunit has essentially one module, the catalytic core, with an insertion; it is the catalytic subunit. The other monomer, the b subunit, is a multidomain subunit; it also contains a class II type core module, although it is truncated and therefore inactive. In addition, this subunit possesses several additional domains: two similar domains that contain helix-turn-helix motifs characteristic of some DNA-binding proteins, a domain that is similar to the anticodon-binding N-terminal domain of aspartyl-tRNA synthetase, a domain that is similar to some eukaryotic signal transduction proteins, and an RNA-binding domain that resembles the N-terminal domain of the U1A protein (a component of the eukaryotic small nuclear ribonucleoprotein particles that participate in premessenger RNA splicing).

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tRNA-binding Modes of the Two Classes Further Reading

The enzymes of the two classes bind the acceptor arm of tRNA in two ways that constitute mirror images of each other (Figure 2). Class I enzymes bind the tRNA on the side opposite of the variable loop and interact with the minor groove of the acceptor arm double helix. Class II enzymes bind the tRNA on the variable loop side and interact with the major groove of the acceptor arm double helix. This leads to the distinct regiospecicity of the aminoacylation reaction. The mirror symmetrical feature already becomes apparent in the rst step of the reaction (step [I]). Starting with an extended ATP conformation, class I enzymes form bent aminoacyl-adenylates. Class II enzymes form extended aminoacyl-adenylates from ATP in a bent conformation.
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