Small Molecules: Big Players in the Evolution of Protein Synthesis

Jun 16, 2006 - Small Molecules: Big Players in the Evolution of Protein Synthesis. Sandro F. Ataide† and Michael Ibba†,‡,*. †Department of Mic...
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REVIEW

Small Molecules: Big Players in the Evolution of Protein Synthesis Sandro F. Ataide† and Michael Ibba†,‡,* †

Department of Microbiology and ‡Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210

T

he amino acids (aa) required for translation of messenger RNA are delivered to the ribosome esterified to the 3=-ends of tRNAs (Figure 1). The aminoacylation of tRNAs is catalyzed by aminoacyl-tRNA synthetases (aaRSs), which must discriminate their unique cognate pair of aa and tRNA from among the vast number of similar molecules that exist in the cell (1). Cell survival is totally dependent on the correct functioning of aaRSs, and several different strategies have evolved to ensure the accurate recruitment of aa during protein synthesis (2–4). In addition to accurate aa recognition, other approaches adopted by aaRSs to maintain fidelity include editing (also known as proofreading), gene duplication, and the use of alternative biosynthetic pathways (5). In-depth studies of the aaRS family have demonstrated how the chemistry of a particular aa influenced the evolution of these enzymes in the different kingdoms of life. Here, we will review how the requirement for strict aa discrimination during protein synthesis to ensure accurate translation of the genetic code played a role in forging enzymes with dedicated active sites and discuss the additional functions required to prevent degeneracy during decoding (summarized in Figure 2). In the first section, background information about the importance of aaRSs for cell viability is presented, indicating the diversity of these enzymes. A description of the mechanism of aa discrimination used by the different aaRSs follows, presenting different modes for targeting these enzymes. Different strategies used by aaRSs for discrimination are presented, such as secondary sites to prevent infiltration of the genetic code, divergent pathways of aa-tRNA biosynthesis in different organisms, and different aspects of duplication of aaRSs and their implications for the development of better aaRS inhibitors. Aminoacyl-tRNA Synthetases and Translation. The formation of aa-tRNA is a two-step reaction: after binding

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A B S T R A C T The aminoacyl-tRNA synthetases (aaRSs) are responsible for selecting specific amino acids for protein synthesis, and this essential role in translation has garnered them much attention as targets for novel antimicrobials. Understanding how the aaRSs evolved efficient substrate selection offers a potential route to develop useful inhibitors of microbial protein synthesis. Here, we discuss discrimination of small molecules by aaRSs, and how the evolutionary divergence of these mechanisms offers a means to target inhibitors against these essential microbial enzymes.

*To whom correspondence should be addressed. E-mail: [email protected].

Received for review May 14, 2006 and accepted May 22, 2006. Published online June 16, 2006 10.1021/cb0600200k CCC: $33.50 © 2006 by American Chemical Society

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n

ati o

Activation

n

Ac

tio

tiv

a tiv Ac

Activation

particular cognate tRNAs and aa (10–12). The essential role of the aaRSs in translation places a strong selective pressure on the evolution of these enzymes to prevent mistakes during cognate aa-tRNA formation. The recognition of tRNA requires the identification of a unique set of elements, nucleotides, or modified nucleotides at particular positions (13). These so-called identity elements of a tRNA are often placed in the acceptor and anticodon stems, the anticodon loop, and the variable arm of the tRNA. Because of their size and complexity, tRNAs offer sufficiently diverse recognition elements to allow their specific selection by the corresponding aaRS. Distinguishing between structurally related aa and other small molecules is considerably more problematic, and Figure 1. Scheme for the co-translational insertion of an aa in response to a particular occasional errors in substrate selection are unavoidable codon. tRNA is first aminoacylated with cognate aa. Elongation factor EF-Tu binds (14). Consequently, during evolution, certain aaRSs aa-tRNAaa forming the aa-tRNAaa–EF-Tu–GTP ternary complex, which delivers the aatRNAaa to the ribosomal A site when it is occupied by the corresponding codon on the have acquired appended domains that serve to proofread noncognate aa (15). Other strategies to enhance mRNA. the specificity of small molecule discrimination have to the active site, the ␣-carboxylate of the aa attacks the also appeared during aaRS evolution such as gene ␣-phosphate of ATP leading to the formation of an duplication, trans-editing factors, and pre-translational enzyme-bound mixed anhydride (aminoacyl-adenylate modification. Both cognate recognition and noncognate [aa-AMP]) and an inorganic pyrophosphate leaving aa discrimination have had major roles in shaping group; in the second step, the 2=- or 3=-hydroxyl of the aaRSs at the levels of individual residues, modules, and terminal ribose of the corresponding tRNA performs a nucleophilic attack on the aminoacyl-adenylate leading Translation to formation of aa-tRNA and an AMP leaving group (1). The overall two-step reaction is certainly common to all Modification* trans-editing* synthetases, but whether a common mechanism exists for all aaRS is currently unknown (5). The 20 canonical aaRSs, as found in Escherichia coli and eukaryotes, are found in two highly conserved structural groups with 10 Near-cognate members each, classes I and II (6, 7). The class assignsubstrate Ac n tiv sio ati ments of aaRSs with particular aa specificities have u l c on x E been almost completely conserved through evolution; the only known exception is the representation of lysylcis-editing* AaRS duplication* tRNA synthetase (LysRS) in both groups (see below) (8, 9). Specific structural and mechanistic elements define the members of a class (6, 10). AaRSs from class I Excluded by EF-Tu* possess a Rossman dinucleotide binding domain Figure 2. The fate of near-cognate substrates in protein flanked by two signature motifs, HIGH and KMSKS, while synthesis. Pathways in green lead to translation, while class II contains an active site formed by an extended those in red indicate mechanisms by which near-cognate substrates can be excluded. The * denotes pathways that antiparallel ␤-sheet structure characterized by three may be exploitable as drug targets due to corresponding degenerate sequence motifs. differences between bacteria and eukaryotes. Exclusion The aaRSs are believed to have evolved from two indicates substrates unable to bind the active site common ancestors, one from each class, which productively, and activation indicates substrates able to diverged according to the necessity to discriminate enter the aminoacylation pathway. 286

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REVIEW subunit recruitment. These changes are still evident as a major source of heterogeneity in aaRS structures, presenting the potential for specific antimicrobial targeting (16). Amino Acid Discrimination in aaRS Active Aites. The existence of heterogeneity in active site discrimination among aaRSs in the different kingdoms of life mainly consists of variation in aa composition of the active site and its surroundings. An example of active site divergence is seen in a seryl-tRNA synthetase (SerRS) from the archaeon Methanosarcina barkeri which has a zincdependent aa discrimination, while the bacterial type, present in all kingdoms of life, does not require a zinc for aa discrimination (17). A detailed analysis of the different strategies used by aaRSs to discriminate their cognate aa provides the first step toward assessing the use of aaRS as potential drug targets. AaRSs must specifically recognize their cognate aa from among the vast number of small molecules in the cell with similar physical and chemical properties (14). The presence of both D- and L-enantiomers for each aa, precursors from aa biosynthesis, products of aa degradation, and the natural aa together impose a strong selective pressure for a very specific active site, since all have the potential to disrupt translation (18). Initial selection of certain aa and analogues is dependent on significant differences in size, such as between Gly and Trp, or charge when comparing for example Arg and Glu. The discrimination of aa with smaller differences, for example Asp and Asn, is achieved through a network of highly specific interactions during substrate binding (19, 20). Aspartyl-tRNA synthetase (AspRS) takes advantage of the negative charge of Asp and uses mainly electrostatic interactions with two Arg, one Lys, and one His residues to specifically interact with the two carboxylate groups of Asp. The His residue is important in preventing the binding of Asn as shown by structural, biochemical, and theoretical studies (19). In the closely related asparaginyl-tRNA synthetase (AsnRS), the Lys conserved in the active site of AspRS is replaced by a Gly or Leu and the critical His is absent, allowing preferential binding of Asn rather than Asp (20). Interestingly, asparagine synthase B shares the same binding residues as yeast AspRS; however, Asp binds to the active site in a reverse orientation in order to activate the ␤-carboxylate with AMP instead of the ␣-carboxylate as in AspRS (21). The AspRS/AsnRS discrimination mode illustrates how the expansion of the genetic code to accommodate both www.acschemicalbiology.org

Asp and Asn was facilitated by divergent evolution from an ancestral enzyme to generate two aaRSs with high substrate specificities. The discrimination between Glu and Gln relies on the same principle. The aa specificity was demonstrated by replacing the residues required for Gln binding for the residues required for Glu binding in human glutaminyl-tRNA synthetase (GlnRS). The new enzyme was able to activate and attach Glu to tRNAGln, demonstrating how active site specificity could be modified among related aaRSs (22). In most cases, the fidelity of aa selection is achieved by a network of H-bond and hydrophobic interactions between the aa and the cognate aaRS. Tyrosyl-tRNA synthetase (TyrRS) is the best characterized example of how aaRSs achieve fidelity in aa discrimination (reviewed in ref 23). TyrRS uses an extensive H-bonding network to discriminate Tyr from Phe, and the Y34F replacement disrupts the H-bond network within the active site reducing substrate discrimination (24). However, the substitution W126L enhances the discrimination of Tyr over Phe even with a disruption of an H-bond network with Asp176 (25). The fidelity of aa recognition is dependent on the plasticity of the active site of each aaRS to accommodate the side chains without steric clashes between the substrate and the active site residues of the synthetase (26). Generally, each aaRS has evolved to achieve a useful level of specificity without necessarily maximizing discrimination between the cognate aa and cognate analogues (25–28). This was strikingly illustrated in a recent study that detected substrate analogues for 17 aaRSs that could be aminoacylated to cognate tRNA by the wildtype aaRSs (18). Most of the 92 aa analogues found to be substrates were synthetic compounds and likely never exerted a selective pressure on the aaRS to discriminate against them. In addition to aa binding by specific interactions in the active site, aaRSs also increase specificity in recognition by induced fit (29). AaRSs which use this mechanism (GlnRS, TyrRS, arginyl- [ArgRS], [GluRS], histidyl[HisRS], [LysRS1], and threonyl-tRNA synthetase [ThrRS]) rely on communication between distal regions of the protein to sense the binding of the correct substrate in order to recruit the appropriate catalytic residues into a productive position in the active site. ArgRS, GluRS, GlnRS, and LysRS1 share much of the same induced fit mechanism in which the binding of the cognate tRNA is required in order to form a productive active site conforVOL.1 NO.5 • 285–297 • 2006

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O

O H 2N

H2N

OH

S

OH NH2

NH2

S-(2-Aminoethyl)-L-cysteine (AEC)

L-Lysine

H3C

O

NH

NH CH3

H O

OH

N

NH2

NH

O Indolmycin

L-Tryptophan

and used strictly as a eukaryotic ThrRS inhibitor in clinical N CH3 O studies against malaria and O O O N N O O H3 C antiangiogenesis (42, 43). O P O CH3 O H 3C OH O OH NH2 The mode of borrelidin inhibiOH OH OH OH OH H 3C tion is by binding to a hydrophobic noncatalytic domain, Mupirocin (Pseudomonic acid) L-Isoleucyl-adenylate cluster A, which impairs catalytic conformational changes HO OH O NH2 O O in ThrRS, resulting in reduced O P NH OH N N binding of ATP and Thr (44). N OH N O OH N O N H3 C The antiangiogenesis action O OH N O N O P O O H C of borrelidin is due to inhibi3 CH3 NH2 NH P O OH O HO tion of ThrRS and activation of OH CH OH OH OH 3 caspases 3 and 8 to induce L-Leucyl-adenylate Agrocin 84 apoptosis (42). Indolmycin is a potent NH2 NH2 bacterial TrpRS inhibitor that N N N N acts as a competitive inhibitor N N O N O N due to a differential binding to O- P O tRNA O P O O O the enzyme compared to Trp ONH3+ ONH3+ (Figure 3) (38, 45). Since OH HN OH O CH3 CH3 indolmycin is a biosynthetic O O derivative of Trp, it has other 2'-(L-Norvalyl)amino-2'-deoxyadenosine (Nva2AA) 2'-(L-Norvaline)ester-adenosine (2'-ester Nva-tRNALeu) mechanisms of action inside the cell which cumulatively Figure 3. Chemical structures of cognate substrates and inhibitors of aaRSs. affect viability (46, 47). Unfortunately, the inhibitory action is not widespread among mation and trigger aa activation (19, 30–35). In these pathogens, probably due to its hydrophobicity that examples ATP and cognate aa binding are enhanced impairs uptake by certain organisms. Although indolupon recognition of the correct tRNA by the anticodon mycin is commercially available for research only, it is binding region. Furthermore, in GlnRS, binding of not FDA-approved. Indolmycin acts as a bacteriostatic cognate tRNA is enhanced if the cognate aa (Gln) is bound to the active site, while the near-cognate aa (Glu) agent against Staphylococcus aureus, which can acquire reduces tRNA binding 60-fold (31). In ThrRS and TyrRS, elevated resistance against indolmycin via a point mutainduced fit upon binding of the correct substrate leads tion that causes the H43N replacement in TrpRS (48). A directly to formation of a productive active site (36, 37). bacteriocidal effect of indolmycin was observed against Helicobacter pylori, which was unable to develop resisThe indication that induced fit serves as an extra discrimination factor in binding of both cognate aa and tance (49). Also, in vitro and in vivo studies in Streptomyces griseus, which produces indolmycin, revealed a cognate tRNA demonstrates how aaRSs have evolved second copy of TrpRS that confers the resistance to different mechanisms for selection of the correct indolmycin (45). substrate. Of the numerous aaRS inhibitors, only mupirocin A number of known inhibitors of aaRSs act by inter(pseudomonic acid), which inhibits IleRS, is commerfering with recognition of the cognate aa at the active site. The search for aaRS inhibitors has identified many cially available and FDA-approved (50). Structural and biochemical studies indicate the same overall mode of natural products such as indolmycin (inhibitor of binding by mupirocin as the Ile-AMP intermediate tryptophanyl-tRNA synthetase [TrpRS]) (38), borrelidin (Figure 4), the difference being that the inhibitor (inhibitor of ThrRS) (39), and mupirocin (inhibitor of contains a nonanionic acid moiety which fits into an IleRS) (40, 41). Borrelidin is available for research only NH2

N

288

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REVIEW unoccupied hydrophobic pocket in IleRS. The binding of mupirocin in the hydrophobic pocket of IleRS then blocks the subsequent binding of isoleucine and ATP, as confirmed by substitution of the nonanionic acid moiety by a shorter alkyl group that resulted in loss of inhibition (40, 41, 50, 51). Because of the competitive inhibition of IleRS and the bacteriostatic effect, several mupirocin-resistant S. aureus strains were isolated with point mutations in IleRS (16, 41). Most of the resistance against mupirocin derives from the V588F replacement, which was shown to interact with the nonanionic acid moiety. The mode of action of mupirocin illustrates how exploiting noncatalytic motifs and hydrophobic regions around the active sites can effectively inhibit function and may be exploitable for other aaRSs. The most potent competitive inhibitors targeted against any of the aaRSs are variants of the aa-AMP, which is the key intermediate of the aminoacylation reaction (52). All the aminoacyl-adenylates present a KI in the low nanomolar range. Several aa-AMP variants have been synthesized in a nonhydrolyzable form with a sulfamoyl or an aryl-replacement of the ␣-phosphate group of the adenine (53–55). The nonhydrolyzable analogues are potent inhibitors, but the selectivity of these compounds is not restricted to the bacterial aaRSs, and they can potentially also inhibit the host enzyme. Adenylate analogues are widely used to determine X-ray structures of aaRSs and to understand the interactions of the active site with the substrates in mechanistic studies (56). Several compounds that mimic the aa-AMP intermediate have been characterized in screenings of natural products and in larger synthetic library screenings. Of the identified aa-AMP analogues, Agrocin 84 is known to be a biocontrol of plant tumors caused by Agrobacterium tumefaciens and mimics leucyl-adenylate (Figure 3) (57). Agrocin 84 possesses a D-glucofuranosyloxyphosphoryl group linked to the adenine moiety, which is important for the uptake of the compound by the pathogen but must be cleaved to release the toxic moiety which can act as a competitive inhibitor of LeuRS. The toxic moiety has a stable phosphoroamidate bond instead of the labile phosphoroanhydride of the genuine aa-AMP, which makes it a better inhibitor (57). A similar strategy is used by the AspRS inhibitor microcin C, a pentapeptide processed by the cell to generate an Asp-AMP analogue with an N-acyl phosphoroamidate linkage (58). www.acschemicalbiology.org

The variation displayed by bacterial and eukaryotic aaRSs such as aa insertions and variations around the active site are the key features exploited to date when using these enzymes as potential drug targets. Protein sequence alignments and modeling on 3D X-ray structures are not, however, sufficient to predict such sites (17). Details of these variations will only come from a combination of biochemical and structural studies of different aaRS candidates for drug targeting. Also, understanding the different mechanisms of compound uptake by pathogens can lead to a better design of compounds such as Agrocin 84 and microcin C, which have the potential to be used as the basis for the design of highly selective targeted aaRS inhibitors. The design of these compounds will require a better understanding of the physiology of interesting pathogens and their hosts allied with genomics, proteomics, and the screening of compound libraries. Unfortunately, much still remains to be characterized to provide a comprehensive starting point for the rationale design of drugs with the same, targeted, characteristics of Agrocin 84 and microcin C. Amino Acid Discrimination in Editing Sites. As in the case of Asp/AsnRS and Glu/GlnRS, another subgroup of aaRSs also clearly shares common origins, but the degree of similarity between their aa substrates imposes additional constraints on accurate recognition. Discrimination between the aliphatic aa Val, Ile, and Leu (59–62) poses an obvious problem, as the small differences in their potential binding energies preclude the high level of specificity observed for Asp and Asn. The corresponding aaRSs have evolved a proofreading mechanism, named editing, which consists of a secondary site which is able to recognize and hydrolyze aa-AMP and misacylated tRNAs (Table 1). The requirement for an editing site is conserved among the different kingdoms of life and strictly required to maintain cell viability (63). In a few cases, when the editing KEYWORDS domain is absent or inactive in the aaRS, a trans-acting factor with editing activity may come into play. Editing is not, however, ubiquitous; for example, both phenylalanyltRNA synthetase (PheRS) and LeuRS from mitochondria have lost their respective editing

Transfer RNA (tRNA): Small adaptor RNA responsible for delivering amino acids to the ribosome during translation of messenger RNA. Aminoacyl-tRNA Synthetases (aaRS): Family of enzymes responsible for attaching amino acids to the 3=-ends of tRNAs. Amidotransferases: Enzymes responsible for chemical conversion of Glu into Gln or Asp into Asn in a tRNA-dependent manner.

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Figure 4. Visualization of the IleRS structure from Thermus thermophilus with inhibitor (Mupirocin) or nonhydrolyzable adenylated analogue (Ile-AMS, 5=-O-[N-(Lisoleucyl)sulphamoyl]adenosine) bound to the active site (50). Insets indicate the location of the active site on the IleRS structure. The surface representation is transparent blue in order to provide a realistic view of the active site. Color code for stick structures: carbon, yellow; oxygen, red; nitrogen, blue; and sulfur, orange.

activities (64, 65). Several editing activities of aaRSs are presented below, together with examples of cases in which the editing function was lost and a trans-acting factor rescues the activity. IleRS activates noncognate Val only ⬃200-fold less efficiently than cognate Ile, creating a potentially high level of misincorporation of noncognate aa by the ribosome during protein synthesis (66). IleRS corrects this potentially catastrophic misactivation of Val by using an editing mechanism that allows hydrolysis at a secondary active site, named the editing site (67–70). This leads to a substantially lower error rate of ⬍1:3000, compatible with the overall level of fidelity observed in translation. LeuRS (62, 71), valyl- (ValRS) (60), methionyl(MetRS) (72, 73), prolyl- (ProRS) (74), alanyl-tRNA synthetases (AlaRS) (75), ThrRS (76), and PheRS (77, 78) all possess comparable editing activities that minimize the degeneracy of the genetic code by clearing misactivated aa and misacylated tRNAs. The domains responsible for the hydrolysis of misacylated tRNA differ between class I and class II and even within the same class of aaRS (15). Usually, the proofTABLE 1.

AaRS known to possess editing activity and their noncognate substrates

AaRS

Class I IleRS LeuRS

290

Edited noncognate substrates

MetRS ValRS

Ala, ␣-aminobutyrate, Cys, homocysteine, homoserine, Thr, Val Homocysteine, ␥-, ␦-hydroxyisoleucine, ␥-, ␦-hydroxyleucine, Ile, Met, norleucine, norvaline Homocysteine Ala, ␣-aminobutyrate, Cys, Ser, Thr

Class II AlaRS LysRS2 PheRS ProRS ThrRS

Gly, Ser Homocysteine, ornithine Ile, Tyr Ala, Cys Ser

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reading domain excludes binding of the cognate aa-tRNA and binds only the misacylated tRNA. The mechanism by which the connective peptide 1 (CP1) domains of IleRS, ValRS, and LeuRS proofread the misacylated tRNA is by accepting the small noncognate aa in the editing site, while excluding the large cognate aa. Subsequently, the amino group of the noncognate aa interacts with a conserved aspartic acid residue present in the editing site, which is responsible for the correct positioning of the substrate for hydrolysis of the ester bond (71, 79, 80). CP1 is a globular domain positioned ⬃40 Å away from the catalytic site and can be easily accessed by the movement of the CCA end of the tRNA to reposition the misacylated 3=-end into the proofreading site (62). The editing sites of ValRS, LeuRS, and IleRS are present in all kingdoms of life, although LeuRS from human mitochondria has lost the editing function (64). Class II ThrRS possesses another proofreading motif which is believed to hydrolyze the misacylated tRNA using an H2O molecule that is deprotonated by a conserved histidine residue present in the proofreading domain (81). The proofreading domains in ThrRS (76) and AlaRS (75) are distinct modules present at the N-terminal and inserted in the C-terminal portion of the enzymes, respectively, while in PheRS, the active and editing sites are in different subunits (65, 77, 78). ProRS can be found in two versions, one “prokaryotic-like”, which contains the editing domain, while the “eukaryoticlike” version present in human does not edit. This feature is not conserved among all eukaryotic-like ProRS, since the archaeal Methanococcus jannaschii enzyme has editing activity (82). In other instances, additional proteins are recruited in trans to edit misacylated tRNAs as in the case of YbaK and D-Tyr-tRNA deacylase. YbaK specifically hydrolyzes Cys-tRNAPro upon interaction with ProRS, but in a free form is unable to compete with EF-Tu for the misacylated tRNA or even discriminate the correct substrate for deacylation (83–86). Two other forms of deacylases are also known: in bacteria and eukaryotes, D-Tyr-tRNA deacylase hydrolyzes D-Tyr-tRNATyr and also D-Asp-tRNAAsp and Trp D-Trp-tRNA (87–89); in some archaea, a paralog of D-Tyr-tRNA deacylase instead hydrolyzes misacylated Ser-tRNAThr, compensating for the lack of an N-terminal proofreading domain in certain ThrRSs (90, 91). Inhibitory metabolites, or precursors of the cognate aa, which can bypass active and editing site discrimination by aaRSs are good candidates for drug design. For www.acschemicalbiology.org

REVIEW example, ␣-aminobutyrate is a competitive inhibitor of ValRS (63, 92), and norvaline is a competitive inhibitor of LeuRS (93) which can be incorporated during protein synthesis. A series of new synthetic compounds which can be aminoacylated onto the cognate tRNA by aaRSs was reported recently (18), and the different chemical functionalities present in those compounds can be useful to investigate candidates for inhibition of both, active and editing sites. A potential class of inhibitors that has not been investigated are compounds that mimic the noncognate aminoacyl-tRNA form such as 2=-(L-norvalyl)amino-2=-deoxyadenosine (Nva2AA) which inhibits LeuRS (71) (Figure 5), thereby targeting the proofreading domain of a specific aaRS (16). While evolutionary divergence of editing sites confirms these as promising targets, difficulties in synthesizing the equivalent of misacylated tRNA in a stable form and analysis of the specific inhibitory mode and its cellular consequences still need to be investigated. Nevertheless, in many cases, cellular viability necessitates careful proofreading activity, suggesting this class of compounds (63, 94), which would target the proofreading domains of aaRSs, as promising new candidates with a strong potential for drug target development. In addition to these elaborations of aaRSs, other post-aminoacylation pathways have also evolved both to allow code expansion and to provide new aa biosynthetic pathways. Heterogeneity in Alternative aa-tRNA Synthesis. Some bacteria and archaea lack one or more aaRSs, and a tRNA-dependent pathway is used in those organisms to synthesize the cognate aa. Because of the heterogeneity of these pathways and their obligate activity to sustain life, understanding their mechanism of action and developing compounds to target these enzymes is a promising source of drug targets. Many prokaryotes lack AsnRS and/or GlnRS, and in those organisms, the AspRS and/or GluRS are able to mischarge tRNAAsn or tRNAGln with Asp or Glu, respectively. An amidotransferase then converts the Asp-tRNAAsn or Glu-tRNAGln into Asn-tRNAAsn or GlntRNAGln, respectively (Table 2) (95–99). While these enzymes are predominantly microbial, the Glu-tRNAGln amidotransferase is found in chloroplasts and was thought to be present in mitochondria (100). However, it was recently shown that mitochondria instead use GlnRS, and the amidotransferases have become strong candidates to be exploited as bacterial drug targets www.acschemicalbiology.org

(101). The synthesis of initiator fMet-tRNAfMet is also dependent on a similar mechanism in bacteria and organelles (102, 103). Initially, tRNAfMet is aminoacylated with Met by MetRS and further converted by Met-tRNA formylase into fMet-tRNAfMet, which is then used in translation initiation (Table 2) (104). A related method used to ensure the correct charging of the cognate tRNA is the exclusive biosynthesis of a certain aa on the 3=-end of the tRNA. The best known example is the synthesis of selenocysteine-tRNASec, which starts with seryl-tRNA synthetase (SerRS) misacylating tRNASec with serine. Selenocysteine synthase (SelA) then catalyzes the conversion of serine to selenocysteine on tRNASec (105–107). Another recently described example of the synthesis of an aa directly on tRNA involves Cys biosynthesis in certain archaea. In these organisms, a new aaRS named ortho-phosphoseryl-tRNA synthetase (SepRS) first catalyses the attachment of ortho-phosphoserine to tRNACys which is further converted into Cys-tRNACys by the Sep-tRNACys-tRNA synthase (SepCysS) enzyme (108). The overall Sec pathway is universally conserved, including the synthesis of Sec-tRNASec, the decoding of a UGA codon as a Sec, the presence of a Selenocysteine Insertion Sequence (SECIS) element, and the presence of a dedicated EF-Tu homologue (SelB) for delivery of Sec-tRNASec (107). However, among the kingdoms of life, the differences in the Sec pathway can potentially be exploited as drug targets; for example, no homologue of bacterial SelA has yet been found in eukaryotes, and the pathway is believed to be substantially different (109). Also, the SECIS elements of bacterial mRNA are often placed downstream of the AUG site in a coding region, while in eukaryotes and archaea, they are always in the 3= untranslated region (110). The differences in SelB lie in the fact that the bacterial version recognizes and interacts with SECIS through its C-terminal region, KEYWORDS while the eukaryal and Deacylation: Hydrolysis of ester bond between amino acid and 2=- or 3=-hydroxyl of the 3=archaeal forms lack the end of the tRNA. C-terminal domain and Misacylation: Mistake committed by aaRS in instead interact with SECIS attaching a near- or noncognate amino acid to the cognate tRNA. binding protein 2 (SBP2) (111, Editing site: Secondary site in aaRS responsible 112). All these differences in for deacylation of misacylated tRNA. the Sec synthesis and inserGenetic code degeneracy: Error in decoding the genetic code upon incorporation of the wrong tion pathways have the potenamino acid in response to a particular mRNA tial to be exploited as antimicodon. crobial drug targets. VOL.1 NO.5 • 285–297 • 2006

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Figure 5. Visualization of the LeuRS structure from Thermus thermophilus with editing site inhibitor (Nva2AA) and nonhydrolyzable adenylated analogue (Leu-AMS, 5=-O[N-(L-leucyl)sulphamoyl]adenosine) bound to the editing and active site, respectively (71). Insets indicate the location of the editing site at the left and active site at the right side of the LeuRS structure. The surface representation is transparent blue in order to provide a realistic view of the editing and active sites. Color code for stick structures: carbon, yellow; oxygen, red; nitrogen, blue; and sulfur, orange.

is able to charge tRNATrp with 4-nitro-tryptophan and 5-hydroxy-tryptophan (113, 114). The most intriguing case of functional duplication involves LysRS, which can be present either as two versions of class II-type LysRS (LysRS2) with a housekeeping version lysS and a heat-shock-inducible version lysU, or as the structurally unrelated class I-type LysRS (LysRS1) and LysRS2. In organisms that harbor two copies of LysRS2, the second copy (lysU) is expressed under specific conditions such as heat shock and in media containing leucine. The inducible LysRS2 is resistant to inhibition by lysine analogues such as cadaverine, indicating that this duplication of activity is important to avoid misincorporation of lysine analogues under stress conditions (115–117). LysRS1 exists mainly in archaea and some pathogenic bacteria, and this enzyme is more selective and less prone to inhibition when compared to LysRS2 (118). The different versions of LysRS take advantage of different networks of interactions in their active sites to discriminate the correct substrate; where LysRS2 relies on a large network of H-bonding interactions, LysRS1 employs a minimal H-bond interaction in a specific part of the active site. Discrimination against near-cognate analogues which are natural metabolites such as S-(2-aminoethyl)-Lcysteine (AEC), homocysteine, and ornithine can be a serious problem for LysRS2 which does not possess post-transfer editing activity (115, 118). LysRS2 can activate homocysteine and ornithine with ATP, but before they can be transferred to tRNALys, they are further cyclized into homocysteinethiolactone and ornithine lactone, respectively, and released (115). AEC is potentially more problematic since, once activated by LysRS2, it can be transferred to tRNALys and used in protein synthesis by the ribosome, inhibiting cell

The heterogeneity of tRNA-dependent aa synthesis provides many promising targets; however, structural and functional studies must still be performed to understand the potential for inhibition and the cellular consequences of inhibiting these pathways. Compounds targeting the different enzymes involved in amidotransferase, formylase, and selenocysteine synthesis are promising candidates for drug development. In addition to these post-aminoacylation pathways, orthologous and nonorthologous duplication of aaRSs provides another new opening both for drug design and studies of potential antibiotic resistance. Discrimination of Small Molecules through aaRS Duplication. In addition to the various mechanisms described above, some organisms possess a second ortholog of the same aaRS, which often confers resistance to certain conditions or inhibitors by discriminating against small molecules. The existence of such duplicates occurs in organisms exposed to certain inhibitory compounds that, in order to survive, evolved a resistant version of the same aaRS. Although this implies that gene duplication events can be a pitfall for drug targeting, in fact they can also be used as models, since the resistance mechanism can be predicted, studied, and characterized to provide a means to develop better inhibitors that circumvent known problems. TrpRS can be found in duplicate in streptomycetes that produce indolmycin, where only one constitutively expressed copy is sensitive to indolmycin, while the second is expressed to rescue the cell when the inhibitor is synthesized TABLE 2: Noncanonical aminoacyl-tRNA synthesis in translation (45). TrpRS can also be Final product Aminoacyl-tRNA AaRS Substrate for found in Deinococcus for translation radiodurans as two less Asn Asp-tRNA AspRS gatCAB (amidotransferase) Asn-tRNAAsn similar variants, TrpRS1 Cys Sep-tRNA SepRS SepCysS Cys-tRNACys and TrpRS2. In this case, fMet Met-tRNA MetRS Met-tRNA formylase fMet-tRNAfMet the housekeeping Gln Glu-tRNA GluRS gatCAB/gatDE (amidotransferase) Gln-tRNAGln TrpRS1 is resistant to Pyl Pyl-tRNA PylRS Translation Pyl-tRNAPyl inhibition and misacylaSec Ser-tRNA SerRS SelA Sec-tRNASec tion of tRNATrp with Trp analogues, while TrpRS2 292

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REVIEW growth (118). The class I version of LysRS is always less active when compared to the class II version, which seems to be more robust with respect to cognate aminoacylation (27, 28, 118). Interestingly, LysRS1 is not significantly inhibited by AEC either in vitro or in vivo, indicating different mechanisms of recognition and discrimination of lysine and analogues compared to LysRS2 (Figure 3) (118, 119). The fact that AEC is a naturally occurring metabolite (discussed in ref 119), and represents one of the possible compounds which might have exerted a selective pressure in the evolution of two distinct LysRSs with different discrimination of lysine analogues, makes LysRS1 a good candidate to be exploited as an antimicrobial drug target. The requirements of an organism to retain either LysRS1 or LysRS2 are likely based on the necessity for a more selective enzyme versus a more active one, possibly depending on growth physiology and the presence of inhibitors in the environment (27, 28). TyrRS, ThrRS, IleRS, MetRS, and TrpRS can also be found in certain genomes as apparent duplicates, and the expression of the second copy is often dependent upon the presence of inhibitors for the housekeeping version or changes in cellular physiology (45, 120–124). The extensive duplication seen among aaRSs also has important practical implications. Clinical treatment with mupirocin has resulted in the emergence of two resistance mechanisms: high-level resistance, which derives from acquisition of a plasmid-borne IleRS ortholog, or low-level resistance resulting from point mutations at two sites in the gene encoding IleRS (41, 125–127). Studies have demonstrated that the low level of resistance can be reverted by compensatory mutations acquired when the organism is not under antimicrobial pressure (41, 125). Screening of new potent synthetic drugs targeting bacterial MetRS has identified resistant organisms harboring two copies of MetRS. The second copy, which is resistant to the synthetic compounds, possesses an insertion of 27 aa around the active site and is present in several organisms (121). Bioinformatics studies have identified the origin of the second copy of MetRS as deriving from soil-dwelling bacteria which have never been exposed to these synthetic inhibitors (120). These examples summarize how aaRSs can readily adapt and evolve to gain specific functions and avoid degeneracy during decoding. The robust scaffold on which aaRSs are built allows such adaptations to different conditions to which an organism might be exposed, such as the www.acschemicalbiology.org

presence of potent inhibitors. While a drawback in some instances, gene duplication can be exploited to understand the extent of plasticity of aaRSs and characterize the weaknesses of both copies, thereby using this feature of aaRSs to develop new drugs potentially less susceptible to resistance. Conclusions. In recent years, the discovery of new functions and activities among the aaRSs has broadened our knowledge of these essential housekeeping enzymes. Proteomics studies, along with the search for other substrates within the cell, have shown that aaRSs are more versatile than previously believed. In parallel, efforts to expand the genetic code to allow the insertion of unnatural aa have focused on first modifying aaRS specificity (14, 128). On the basis of the often severe loss of activity in many of these modified aaRSs, and studies with inhibitors on the mechanism of substrate recognition, it is becoming clearer how specificity has evolved in these enzymes. Certain aaRSs have apparently evolved to acquire the best balance between activity and specificity, depending on how easily a mistake could be made in misacylating cognate tRNA under particular physiological conditions. Contrary to the common belief that aaRS evolution was primarily driven by tRNA recognition, there has been significant selective pressure to discriminate the cognate aa from other small molecules in the cell. A particularly striking illustration of this comes from the recent discovery of an aaRS specific for the rare amino acid pyrrolysine (129, 130). Understanding the mechanisms by which aaRSs discriminate small molecules provides the opportunity to design drugs that are small molecule inhibitors of specific aaRSs. Characterization of the mechanisms of substrate recognition at the active and editing sites provides a rationale for the development of potent inhibitors for a specific aaRS, which will require structural and biochemical data to improve the design of drugs targeting each of these sites. In parallel, genomics, bioinformatics, and proteomics offer a better potential to identify trans-editing proteins and tRNA-dependent aa biosynthesis and circumvent the most common mode of aaRS resistance, namely, duplication, further enhancing the promise of success for new inhibitors. Acknowledgment: We thank C. Hausmann, J. Ling, and T. E. Rogers for critical reading of the manuscript, and the anonymous reviewers for their helpful and constructive comments. This work was supported by Grant GM 65183 from the National Institutes of Health. S.F.A. is supported by an American Heart Association Predoctoral Fellowship. VOL.1 NO.5 • 285–297 • 2006

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