Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications.

AIMS: The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. METHODS AND RESULTS: The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6.5 and at 50-60 degrees C. Furthermore, EstA remained stable at pH 6-8 and below 50 degrees C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). CONCLUSION: The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.

The many routes of bacterial transfer RNAs after aminoacylation.

Subsequent to their aminoacylation, tRNAs are subject to specific maturation and/or correction processes. Aminoacylated tRNAs ready for use in translation are then specifically channelled to the ribosomal A or P sites. Structural and biochemical studies have opened the way towards furthering our understanding of these routes to the ribosome, which involve a strict distinction between initiator and elongator tRNAs.

Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase.

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated form an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the alpha- and beta-subunits composing the native alpha 2 beta 2 enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon.

Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo.

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.

Control of peptide deformylase activity by metal cations.

Previous work indicated that peptide deformylase behaves as a metalloenzyme since the Escherichia coli enzyme was shown to copurify with a zinc ion. The present study establishes that nickel:enzyme complexes can also be isolated provided that nickel salts were added in the buffers throughout the purification. Similar results were obtained with the deformylases from Thermus thermophilus and Bacillus stearothermophilus. As a result of nickel binding, the catalytic efficiencies of peptide deformylases increased by two to three orders of magnitude with respect to those of the forms previously characterized. Using the model substrate N-formyl-Met-Ala-Ser, kcat/Km values of 5.4, 1.2 and 25 10(4)M-1s-1 could be obtained for the E. coli, T. thermophilus and B. stearothermophilus enzymes, respectively. This value satisfyingly accounts for the deformylation turnover required in the cell. In vitro characterization of the E. coli enzyme shows that zinc can readily substitute for the bound nickel with the catalytic efficiency decreasing to 80 M-1s-1 in turn. Conversely, the activity of the zinc-containing protein can be significantly improved by addition of nickel to the enzymatic assay.

Mapping of the active site zinc ligands of peptide deformylase.

A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of peptide deformylase, including the ligands of the zinc ion. In particular, because zinc is usually coordinate by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed. The functional consequence of the substitutions was assessed by complementation of a fms-null strain with the help of vectors expressing the mutate genes. In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132HEXXH136 motif of peptide deformylase prevented the indicator strain growing. Most enzyme variants were purified to homogeneity in a second step. Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency. The change of Glu88 also led to a significant decrease in catalytic rate. Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency. The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion. Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack. Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of deformylase. This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily. However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that peptide deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.

A new subclass of the zinc metalloproteases superfamily revealed by the solution structure of peptide deformylase.

Escherichia coli peptide deformylase, a member of the zinc metalloproteases family, is made up of an active core domain composed of 147 residues and of an additional and dispensable C-terminal tail of 21 residues. The three-dimensional structure of the catalytic core could be studied by NMR. 1H and 15N NMR resonances assignments were obtained by two-dimensional and three-dimensional heteronuclear spectroscopy. The structure could be calculated using a set of 1015 restraints for the 147 residues of the enzyme. The overall structure is composed of a series of antiparallel beta-strands which surround two perpendicular alpha-helices. The C-terminal helix contains the HEXXH motif, which is crucial for activity. This helical arrangement and the way the histidines bind the zinc ion clearly are structurally reminiscent of the other members of the metalloprotease family, such as thermolysin or metzincins. Nevertheless, the overall arrangement of secondary and tertiary structures of peptide deformylase and the positioning of its third zinc ligand (a cysteine) are quite different from those of the other members of the family. These discrepancies, together with several biochemical differences, lead us to propose that peptide deformylase is the first example of a new class of the zinc-metalloproteases family. Studies of the interaction of peptide deformylase with either an inhibitor of the reaction or a product of the catalysed reaction, Met-Ala-Ser, as well as comparisons with the structures of other enzymes of the family, have enabled us to delineate the area corresponding to their binding site. The structural basis of the specificity of recognition of the formyl group is discussed in the context of the protease superfamily.

Methionyl-tRNA synthetase zinc binding domain. Three-dimensional structure and homology with rubredoxin and gag retroviral proteins.

Methionyl-tRNA synthetase from Escherichia coli contains one tightly bound zinc atom per subunit. The region encompassing residues 138 to 163 of this enzyme is responsible for the metal binding. A 28-mer peptide corresponding to these residues was expressed in vivo and shown to contain approximately 1 mol of tightly bound Zn/mol of peptide. In this study, the three-dimensional solution structure of this peptide was solved by means of two-dimensional proton NMR spectroscopy. A total of 133 nuclear Overhauser effect distance constraints and 22 dihedral angle restraints were used for the calculations, using a hybrid distance-geometry-simulated annealing strategy. Excluding the first four residues, the resulting structure is well-defined (r.m.s.d. 0.71 A for backbone atoms) and composed of a series of four tight turns. The second and the fourth turns are composed of CXXC sequences which are structurally homologous to the NH-S turns found in the metal binding sites of gag retroviral proteins and rubredoxin. The solution structure of the zinc binding peptide shows significant discrepancies with the crystal structure of methionyl-tRNA synthetase.

Dual level control of the Escherichia coli pheST-himA operon expression. tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli have established that the pheST operon transcription is controlled by a Phe-tRNA(Phe)-mediated attenuation mechanism. More recently, the himA gene, encoding the alpha-subunit of integration host factor, was recognized immediately downstream from pheT, possibly forming part of the same transcriptional unit. By using the in-vitro transcription and S1 mapping techniques, transcription termination after pheT could be excluded, indicating that himA can be expressed from polycistronic messenger RNAs encompassing the pheST region. However, the presence of a secondary promoter able to express himA and located within pheT is demonstrated. To further investigate the regulation of the pheST-himA operon expression, genetic fusions between various parts of this operon and the lacZ gene were constructed and studied. Our results confirm the autoregulation of himA previously described, and demonstrate that it occurs through the modulation of the secondary promoter activity within pheT. Surprisingly, it is found that the pheST promoter is also submitted to the same control. Consistent with this, DNA sequences homologous to the integration host factor binding site consensus are present at the level of both promoters. However, evidence in favor of two different repressor complexes is provided. Previously observed SOS induction of the himA expression is shown to occur through the modulation of both promoter activities. Contrasting with the other genes under SOS control, the LexA protein binding site consensus sequence could not be found in the two promoter regions. This suggests that either the LexA protein directly participates in the formation of an active holorepressor, or that the product of an SOS gene is able to inhibit the formation or the binding of such a repressor. Finally, our results indicate that the pheST-himA operon expression is controlled by two different mechanisms acting independently. (1) The phenylalanyl-tRNA synthetase and the himA product expressions are controlled by an operator-repressor type mechanism, in which the himA product and the SOS network are involved. (2) Through its partial cotranscription with pheST, himA expression is also under attenuation control. The latter control may provide a way to couple the intracellular concentration of the himA product to the functional state of the translational apparatus.

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase.

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).

Structure-function relationships within the peptide deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion.

Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.

Solution structure of the anticodon-binding domain of Escherichia coli lysyl-tRNA synthetase and studies of its interaction with tRNA(Lys).

A protein domain corresponding to residues 31 to 149 of the E. coli Lysyl-tRNA synthetase species corresponding to the lysS gene was expressed and 15N-labelled. 1H and 15N NMR resonance assignments for this domain were obtained by two-dimensional and three-dimensional homonuclear and heteronuclear spectroscopy. Using distance geometry and simulated annealing, a three-dimensional structure could be calculated using 701 NOE and 86 dihedral angle restraints. It is composed of a five-stranded antiparallel beta-barrel capped by three alpha-helices at its ends. This structure closely resembles that of the N-terminal domain of the other E. coli lysyl-tRNA synthetase species expressed from the lysU gene and is highly homologous to the fold observed for the corresponding region of aspartyl-tRNA synthetase. It is shown that the isolated N-terminal fragment of lysyl-tRNA synthetase can interact with tRNA(Lys) as well as with poly (U), which mimics the anticodon sequence. Amino acid residues involved in these interactions were identified and, in the case of poly-U, a number of specific protein-RNA contacts were characterized. Specific recognition of tRNA(Lys) involves a cluster of four structurally well-defined aromatic residues, anchored on the beta-strands, and basic residues located on the surrounding loops. This organization is reminiscent of other RNA binding proteins, such as the U1A small nuclear ribonucleoprotein.

Methionyl-tRNA synthetase needs an intact and mobile 332KMSKS336 motif in catalysis of methionyl adenylate formation.

The family of aminoacyl-tRNA synthetases may be split into two classes according to the occurrence of specific combinations of peptide motifs. This study deals with the functional role of the KMSKS motif, which, in association with the HIGH motif, defines class 1 aminoacyl-tRNA synthetases. Each residue in the 332KMSKS336 sequence of Escherichia coli methionyl-tRNA synthetase, as well as R337 and the two surrounding G330 and G338 residues, were mutagenized. The comparison of the kinetic and equilibrium parameters of the methionine activation reaction sustained by the resulting variants enables the following conclusions to be drawn. (1) Mutation of all the residues studied strongly destabilizes the transition state for the formation of methionyl adenylate whilst changing moderately the stability of the ground state ternary complex enzyme, methionine: ATP-Mg2+. The consequences of the mutations are also reflected at the level of the stability of the ground state enzyme, methionyl adenylate:PPi-Mg2+ complex which is systematically decreased. (2) The substitution with alanine of any one of the three basic residues K332, K335 and R337 destabilizes the transition state by more than 3.2 kcal/mol, while substitution of the non-basic residues M333, S334 or S336 destabilizes it by at most 2.5 kcal/mol. Such a difference may reflect different modes of action of the residues, with the basic ones directly interacting with the beta and gamma phosphates of the ATP-Mg2+ substrate and the non-basic ones playing a structural role and/or participating in mobility of the enzyme region carrying the motif. (3) Modification of G330 or G338 to a proline markedly decreases the kinetic rate of methionyl adenylate formation. This behaviour suggests that the flexibility of the KMSKS loop in the structure of methionyl-tRNA synthetase is required to reach the transition state during formation of methionyl adenylate.

Mapping of the zinc binding domain of Escherichia coli methionyl-tRNA synthetase.

Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms. Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms. In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E. coli methionyl-tRNA synthetase were successively changed into an alanine. Either substitution is sufficient to destabilize the tight binding of the zinc ion. Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared. It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate. These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc. In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack. The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.

Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity.

Escherichia coli strains with abnormally high concentrations of bis(5'-nucleosidyl)-tetraphosphates (Ap4N) were constructed by disrupting the apaH gene that encodes Ap4N-hydrolase. Variation deletions and insertions were also introduced in apaG and ksgA, two other cistrons of the ksgA apaGH operon. In all strains studied, a correlation was found between the residual Ap4N-hydrolase activity and the intracellular Ap4N concentration. In cells that do not express apaH at all, the Ap4N concentration was about 100-fold higher than in the parental strain. Such a high Ap4N level did not modify the bacterial growth rate in rich or minimal medium. However, while, as expected, the ksgA- and apaG- ksgA- strains stopped growing in the presence of this antibiotic at 600 micrograms/ml. The were not sensitive to kasugamycin, the apaH- apaG- ksgA- strain filamented and stopped growing in the presence of this antibiotic at 600 micrograms/ml. The growth inhibition was abolished upon complementation with a plasmid carrying an intact apaH gene. Trans addition of extra copies of the heat-shock gene dnaK also prevented the kasugamycin-induced filamentation of apaH- apaG- ksgA- strains. This result is discussed in relation to the possible involvement of Ap4N in cellular adaptation following a stress.

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.

Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition.

Site-directed nuclease digestion and nonsense mutations of the Escherichia coli metG gene were used to produce a series of C-terminal truncated methionyl-tRNA synthetases. Genetic complementation studies and characterization of the truncated enzymes establish that the methionyl-tRNA synthetase polypeptide (676 residues) can be reduced to 547 residues without significant effect on either the activity or the stability of the enzyme. The truncated enzyme (M547) appears to be similar to a previously described fully active monomeric from of 64,000 Mr derived from the native homodimeric methionyl-tRNA synthetase (2 x 76,000 Mr) by limited trypsinolysis in vitro. According to the crystallographic three-dimensional structure at 2.5 A resolution of this trypsin-modified enzyme, the polypeptide backbone folds into two domains. The former, the N-domain, contain a crevice that is believed to bind ATP. The latter, the C-domain, has a 28 C-residue extension (520 to 547), which folds back, toward the N-domain and forms an arm linking the two domains. This study shows that upon progressive shortening of this C-terminal extension, the enzyme thermostability decreases. This observation, combined with the study of several point mutations, allows us to propose that the link made by the C-terminal arm of M547 between its N and C-terminal domains is essential to sustain an active enzyme conformation. Moreover, directing point mutations in the 528-533 region, which overhangs the putative ATP-binding site, demonstrates that this part of the C-terminal arm participates also in the specific complexation of methionyl-tRNA synthetase with its cognate tRNAs.

Two acidic residues of Escherichia coli methionyl-tRNA synthetase act as negative discriminants towards the binding of non-cognate tRNA anticodons.

Escherichia coli methionyl-tRNA synthetase recognizes its cognate tRNAs according to the sequence of the CAU anticodon. In order to identify residues of methionyl-tRNA synthetase involved in tRNA anticodon recognition, enzyme variants created by cassette mutagenesis were genetically screened for their acquired ability to charge tRNA(mMet) derivatives with an ochre or an amber anticodon and, consequently, to cause the suppression of a stop codon in an indicator gene. The selected enzymes are called suppressors. Mutations were firstly directed towards the region of the synthetase encompassing residues 451 to 467. Several dozens of suppressor enzymes were compared. Statistical analysis of the mutations suggested that the substitution of an Asp side-chain at position 456 was sufficient to render possible the charging of the ochre or amber suppressor tRNAs. Point mutants at this position were therefore constructed. Their behaviour demonstrated that various tRNA(Met) derivatives having a non-Met anticodon could be aminoacylated in vitro provided only that the side-chain of residue 456 was no longer acidic. In turn, the Asp456 residue is not essential to the CAU anticodon recognition, since its substitution does not impair the aminoacylation of wild-type tRNA(Met). The analysis was enlarged to a second region from residue 437 to residue 454. The mutagenesis highlighted two other positions, one of which, Asn452, appeared involved in wild-type tRNA(Met) binding. The second position, Asp449, plays a role very similar to that of Asp456. It is concluded that both Asp449 and 456 behave as "antideterminants", contributing together to the rejection by the enzyme of tRNAs carrying non-Met anticodons. Finally, it is shown that the activities of some particular methionyl-tRNA synthetase variants, which have been made indifferent to the sequence of the anticodon of a tRNA(Met), are tightly dependent on the presence of the nucleotide determinants specific to the acceptor stem of tRNA(Met).

Solution structure of nickel-peptide deformylase.

In the accompanying paper, we report that zinc is unlikely to be the co-factor supporting peptide deformylase activity in vivo. In contrast, nickel binding promotes full enzyme activity. The three-dimensional structure of the resulting nickel-containing peptide deformylase (catalytic domain, residues 1 to 147) was solved by NMR using a 13C-15N-doubly labelled protein sample. A set of 2261 restraints could be collected, with an average of 15.4 per amino acid. The resolution, which shows a good definition for the position of most side-chains, is greatly improved compared to that previously reported for the zinc-containing, inactive form. A comparison of the two stuctures indicates however that both share the same 3D organization. This shows that the nature of the bound metal is the primary determinant of the hydrolytic activity of this enzyme. Site-directed mutagenesis enabled us to determine the conserved residues of PDF involved in the structure of the active site. In particular, a buried arginine appears to be critical for the positioning of Cys90, one of the metal ligands. Furthermore, the 3D structure of peptide deformylase was compared to thermolysin and metzincins. Although the structural folds are very different, they all display a common structural motif involving an alpha-helix and a three-stranded beta-sheet. These conserved structural elements build a common scaffold which includes the active site, suggesting a common hydrolytic mechanism for these proteases. Finally, an invariant glycine shared by both PDF and metzincins enables us to extend the conserved motif from HEXXH to HEXXHXXG.

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases.

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.