Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNA(Val)-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3'-adenosine of tRNA(Val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNA(Val) is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2'-OH, substitution of the 3'-terminal adenosine of tRNA(Val) with 3'-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3'-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.     

Transiently misacylated tRNA is a primer for editing of misactivated adenylates by class I aminoacyl-tRNA synthetases

The genetic code depends on amino acid fine structure discrimination by aminoacyl-tRNA synthetases. For isoleucyl- (IleRS) and valyl-tRNA synthetases (ValRS), reactions that hydrolyze misactivated noncognate amino acids help to achieve high accuracy in aminoacylation. Two editing pathways contribute to aminoacylation fidelity: pretransfer and post-transfer. In pretransfer editing, the misactivated amino acid is hydrolyzed as an aminoacyl adenylate, while in post-transfer editing a misacylated tRNA is deacylated. Both reactions are dependent on a tRNA cofactor and require translocation to a site located approximately 30 A from the site of amino acid activation. Using a series of 3'-end modified tRNAs that are deficient in either aminoacylation, deacylation, or both, total editing (the sum of pre- and post-transfer editing) was shown to require both aminoacylation and deacylation activities. These and additional results with IleRS are consistent with a post-transfer deacylation event initiating formation of an editing-active enzyme/tRNA complex. In this state, the primed complex processively edits misactivated valyl-adenylate via the pretransfer route. Thus, misacylated tRNA is an obligatory intermediate for editing by either pathway.     

On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction. Analysis of the reaction mechanism by total rate equations

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex. Spermidine.ATP is a weaker substrate. The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.     

Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination. It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase. Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation. Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues. Nevertheless, we isolated an intragenic suppressor from a combinatorial library. The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate. Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex. Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.     

Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases

Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Archaebacterial phenylalanyl-tRNA synthetase. Accuracy of the phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri, Zn(II)-dependent synthesis of diadenosine 5',5'-P1,P4-tetraphosphate, and immunological relationship of OFFnylalanyl-tRNA synthetases from different urkingdoms

Phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri activates a number of phenylalanine analogues (methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid and ochratoxin A) in the absence of tRNA, as demonstrated by Km and kcat of the ATP/PPi exchange reaction. Upon complexation with tRNA, AMP formation from the enzyme X tRNA complex in the presence of ATP, one of the above analogues or tyrosine, leucine, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine indicates activation of the analogues under conditions of aminoacylation. Natural noncognate amino acids are not transferred to tRNAPhe-C-C-A or tRNAPhe-C-C-A-(3'-NH2). This pretransfer proofreading mechanism, together with the comparatively low ratio of synthetic to successive hydrolytic steps, resembles the mechanism of liver enzymes of vertebrates. In contrast, eubacterial phenylalanyl-tRNA synthetases achieve the necessary fidelity by post-transfer proofreading, a corrective hydrolytic event after transfer to tRNAPhe. Diadenosine 5',5'-P1,P4-tetraphosphate synthesis is shown to be a common feature for phenylalanyl-tRNA synthetases from all three lineages of descent. The immunological approach demonstrates that aminoacyl-tRNA synthetases do not belong to the group of enzymes in gene expression with high structural conservation.     

Levels of aminoacyl-tRNA synthetases, tRNA nucleotidyltransferase and ATP in germinating lupin seeds

Transfer RNAs in dry lupin seeds are aminoacylated to a low extent (Kedzierski, W. and Pawe?kiewicz, J. (1977) Phytochemistry 16, 503-504) and are partly degraded at the acceptor terminus (Dziegielewski, T. and Pawe?kiewicz, J. (1977) Bull. Acad. Polon. Sci. Ser. Biol. 7, 4oo-435). Increase in the levels of tRNA aminoacylation and disappearance of defective tRNA molecules during seed germination are not accompanied by significant changes in the levels of phenylalanyl-, arginyl-, valyl-tRNA synthetases and tRNA nucleotidyltransferase. Additionally, no inhibitor of aminoacylation of valine tRNA has been detected in dry seeds. However, dry seeds contain very low ATP amounts, which increase dramatically during germination. The above results suggest that a very low ATP level is a factor limiting the aminoacylation and reparation of tRNA molecules at early stages of seed germination.     

Marcaine, a selective inhibitor of eucaryotic aminoacylation.

The effects of marcaine, a myotoxic drug, on the aminoacylation of transfer ribonucleic acid (rRNA) have been studied. The drug is a potent inhibitor of the acylation of rat liver tRNA with leucine and isoleucine but is only mildly inhibitory (or not inhibitory) to acylation with a number of other amino acids which were tested. Further, marcaine inhibited aminoacylation in cell-free systems using components from several mammalian tissues, including muscle, from yeast, and from wheat germ. No effect of the drug was observed in aminoacylation systems from several bacterial species which were tested. The drug inhibits acylation with leucine and isoleucine competitively but exhibited noncompetitive kinetics when the concentrations of adenosine 5'-triphosphate (ATP) and tRNA were varied. Marcaine was also a competitor of leucine in the ATP--pyrophosphate exchange reaction. Two structural analogues of marcaine, carbocaine and xylocaine, also inhibited acylation of rat liver tRNA with leucine but in a noncompetitive fashion. On a molar basis, marcaine appears to be the most effective inhibitor of the three drugs tested.     

Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules

The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.     

Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.     

Correlating amino acid conservation with function in tyrosyl-tRNA synthetase

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.     

Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation

The selective and potent inhibition of mitochondrial translation in Saccharomyces cerevisiae by pentamidine suggests a novel antimicrobial action for this drug. Electrophoresis mobility shift assay, T1 ribonuclease footprinting, hydroxyl radical footprinting and isothermal titration calorimetry collectively demonstrated that pentamidine non-specifically binds to two distinct classes of sites on tRNA. The binding was driven by favorable entropy changes indicative of a large hydrophobic interaction, suggesting that the aromatic rings of pentamidine are inserted into the stacked base pairs of tRNA helices. Pentamidine binding disrupts the tRNA secondary structure and masks the anticodon loop in the tertiary structure. Consistently, we showed that pentamidine specifically inhibits tRNA aminoacylation but not the cognate amino acid adenylation. Pentamidine inhibited protein translation in vitro with an EC(50) equivalent to that binds to tRNA and inhibits tRNA aminoacylation in vitro, but drastically higher than that inhibits translation in vivo, supporting the established notion that the antimicrobial activity of pentamidine is largely due to its selective accumulation by the pathogen rather than by the host cell. Therefore, interrupting tRNA aminoacylation by the entropy-driven non-specific binding is an important mechanism of pentamidine in inhibiting protein translation, providing new insights into the development of antimicrobial drugs.     

Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid.

The shape of the time curve for the aminoacylation of tRNA has been investigated using five different amino acid:tRNA ligases. Four of these enzymes showed a lag in the time curve during the early phase of the first catalytic turnover of the enzyme. In each case, the lag period could be abolished by preincubating the ligase with amino acid, ATP, and Mg2+ under conditions known to give an aminoacyl adenylate-enzyme complex. With all five ligases the steady state rate of transfer from the preformed aminoacyl-adenylate complex to tRNA was approximately the same as that of the overall reaction.     

Tyrosyl-tRNA synthetase from baker's yeast. Order of substrate addition, discrimination of 20 amino acids in aminoacylation of tRNATyr-C-C-A and tRNATyr-C-C-A(3'NH2)

The order of substrate addition to tyrosyl-tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead-end inhibitors. The kinetic patterns are consistent with a random bi-uni uni-bi ping-pong mechanism. Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate. Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr-C-C-A. The lowest values are observed for Cys, Phe, Trp (D = 28,000-40,000), showing that, at the same amino acid concentrations, tyrosine is 28,000-40,000 times more often attached to tRNATyr-C-C-A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D greater than 500,000); D values of the other amino acids are in the range of 100,000-500,000. Lower specificity is observed in aminoacylation of the modified substrate tRNATyr-C-C-A(3'NH2) (D1 = 500-55,000). From kinetic constants and AMP-formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr-C-C-A hydrolytic proof-reading factors could be calculated for a pretransfer (II 1) and a post-transfer (II 2) proof-reading step. The observed values of II 1 = 12-280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (II 2 = 1-2). Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X-ray analysis of the enzyme from Bacillus stearothermophilus.     

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation.

Seryl tRNA synthetase from Saccharomyces Carlsbergensis C836 contains two sets of sites for tRNASer, L-serine, and Mg2+-ATP, both of which are involved in aminoacylation. This is based on the following experimental results: (a) at low serine concentrations, second order kinetics in tRNASer are observed; (b) biphasic kinetics result when the amino acid is the varied substrate indicating anticooperative binding of two serine molecules to the synthetase; (c) when two molecules of serine are bound the rate of aminoacylation increases strongly and becomes first order in tRNASer; (d) the involvement of more than one site for Mg2+ and ATP is deduced from systematic variations of the concentrations of Mg2+ and ATP. Implications of the anticooperative binding of the substrates for possible reaction mechanisms are discussed. The results indicate that under normal conditions, the activity of seryl tRNA synthetase is regulated mainly by tRNASer while at high serine concentrations regulation by the amino acid itself prevails.     

Variant of human enzyme sequesters reactive intermediate

In cellular environments, coupled hydrolytic reactions are used to force efficient product formation in enzyme-catalyzed reactions. In the first step of protein synthesis, aminoacyl-tRNA synthetases react with amino acid and ATP to form an enzyme-bound adenylate that, in the next step, reacts with tRNA to form aminoacyl-tRNA. The reaction liberates pyrophosphate (PP(i)) which, in turn, can be hydrolyzed by pyrophosphatase to drive efficient aminoacylation. A potential polymorphic variant of human tryptophanyl-tRNA synthetase is shown here to sequester tryptophanyl adenylate. The bound adenylate does not react efficiently with the liberated PP(i) that normally competes with tRNA to resynthesize ATP and free amino acid. Structural analysis of this variant showed that residues needed for binding ATP phosphates and thus PP(i) were reoriented from their conformations in the structure of the more common sequence variant. Significantly, the reorientation does not affect reaction with tRNA, so that efficient aminoacylation is achieved.     

Valylation of the two RNA components of turnip-yellow mosaic virus and specificity of the tRNA aminoacylation reaction.

A comparative study of the aminoacylation of the two RNA components of turnip yellow mosaic virus, of yeast tRNAVal, tRNAfMet and of tRNAPhe by purified yeast valyl-tRNA synthetase is reported. Aminoacylations were performed in the presence of pure yeast tRNA nucleotidyltransferase, since 85% of the viral RNA molecules lacked the 3'-adenosine. We find that aminoacylation of the viral RNAs, like tRNA aminoacylation, reflects an equilibrium between the acylation and deacylation reactions. The kinetic parameters of TYM virus RNA valylation resemble the values found for tRNAVal valylation; in particular, there is a strong affinity between the viral RNA and valyl-tRNA synthetase and the rate constant for TYM virus RNA valylation is only slightly lower than that for tRNAVal. This result contrasts with the reduced rates observed in tRNA mischarging, and suggests that the viral RNA could be easily aminoacylated in vivo. Considering the fact that the 3'-terminal sequence of TYM virus RNA has only a few points of resemblance to a tRNA sequence, we propose that there are some structural motifs found in both tRNAVal and TYM virus RNA which are brought in a similar spatial arrangement recognized by valyl-tRNA synthetase.     

Histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium. Interaction with substrates and ATP analogues.

Structural requirements for substrate binding to histidyl-tRNA synthetase from Salmonella typhimurium have been investigated using ATP analogues. Ki values and the relative binding affinity of the enzyme for these analogues have been determined in the tRNA aminoacylation reaction. The enzyme is highly specific for ATP: no binding was found for GTP, CTP, TTP and UTP. dATP is a very poor substrate for acylation of tRNA, with a Km 40-fold higher than that of ATP. Binding of adenosine 5'-triphosphate requires interactions of the amino group of adenosine and the sugar moiety; the 2' and the 5' positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP is a noncompetitive inhibitor with ATP. The interaction of histidyl-tRNA synthetase, a dimeric enzyme, with histidine and ATP was examined by fluorescence measurements at equilibrium and by equilibrium dialysis. Binding with L-histidine is significantly tighter at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 7.5 by equilibrium dialysis and is 1 mol ATP/mol enzyme and, variably, close to 2 or 1 mol histidine/mol enzyme.     

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.