Obtaining New Candidate Peptides for Biological Anticancer Drugs from Enzymatic Hydrolysis of Human and Bovine Hemoglobin.

Enzymatic hydrolysis of bovine and human hemoglobin generates a diversity of bioactive peptides, mainly recognized for their antimicrobial properties. However, antimicrobial peptides stand out for their ability to specifically target cancer cells while preserving rapidly proliferating healthy cells. This study focuses on the production of bioactive peptides from hemoglobin and evaluates their anticancer potential using two distinct approaches. The first approach is based on the use of a rapid screening method aimed at blocking host cell protein synthesis to evaluate candidate anticancer peptides, using Lepidium sativum seed germination as an indicator. The results show that: (1) The degree of hydrolysis (DH) significantly influences the production of bioactive peptides. DH levels of 3 to 10% produce a considerably stronger inhibition of radicle growth than DH 0 (the native form of hemoglobin), with an intensity three to four times greater. (2) Certain peptide fractions of bovine hemoglobin have a higher activity than those of human hemoglobin. (3) The structural characteristics of peptides (random coil or alpha helix) play a crucial role in the biological effects observed. (4) The α137-141 peptide, the target of the study, was the most active of the fractions obtained from bovine hemoglobin (IC50 = 29 ± 1 µg/mL) and human hemoglobin (IC50 = 48 ± 2 µg/mL), proving to be 10 to 15 times more potent than the other hemoglobin fractions, attributed to its strong antimicrobial potential. The second approach to assessing anticancer activity is based on the preliminary in vitro analysis of hydrolysates and their peptide fractions, with a focus on the eL42 protein. This protein is of major interest due to its overexpression in all cancer cells, making it an attractive potential target for the development of anticancer molecules. With this in mind, astudy was undertaken using a method for labeling formylase (formyl-methionyl-tRNA transformylase (FMTS)) with oxidized tRNA. This approach was chosen because of the similarities in the interaction between formylase and the eL42 protein with oxidized tRNA. The results obtained not only confirmed the previous conclusions but also reinforced the hypothesis that the inhibition of protein synthesis plays a key role in the anticancer mechanism of these peptides. Indeed, the data suggest that samples containing α137-141 peptide (NKT) and total hydrolysates may have modulatory effects on the interaction between FMTS and oxidized tRNA. This observation highlights the possibility that the latter could influence molecular binding mechanisms, potentially resulting in a competitive situation where the ability of substrate tRNA to bind efficiently to ribosomal protein is compromised in their presence. Ultimately, these results suggest the feasibility of obtaining candidate peptides for biological anticancer drugs from both human and bovine hemoglobin sources. These scientific advances show new hope in the fight against cancer, which affects a large number of people around the world.

Ribosomal protein eL42 contributes to the catalytic activity of the yeast ribosome at the elongation step of translation.

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteins containing amino acid substitutions within or adjacent to the GGQ minidomain failed to complement the function of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects. These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo. Furthermore, we show that some of the mutations associated with the conserved GGQ region lead to reduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect to puromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for the side chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, our findings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promoting the course of the elongation cycle.

Affinity labelling in situ of the bL12 protein on E. coli 70S ribosomes by means of a tRNA dialdehyde derivative.

In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribosomes; (ii) the stoichiometry of labelling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E. coli bL12 protein is one molecule of PLP covalently bound per polypeptide chain; (vii) the amino acid residue of recombinant bL12 cross-linked with PLP is Lys-65; (viii) Lys-65 of E. coli bL12 corresponds to Lys-53 of eL42 which was previously shown to cross-link with P-site bound tRNAox on human 80S ribosomes in situ; (ix) finally, E. coli bL12 and human eL42 proteins display significant primary structure similarities, which argues for evolutionary conservation of these two proteins located at the tRNA-CCA binding site on eubacterial and eukaryal ribosomes.

A Functional Role for the Monomethylated Gln-51 and Lys-53 Residues of the 49GGQTK53 Motif of eL42 from Human 80S Ribosomes.

BACKGROUND: We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA. We have also recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions. METHOD: Here, we have studied the interactions between recombinant eL42 and eRF1 proteins and the tRNA substrate by means of the Biacore assay, using the wild-type eL42 protein, the eL42-Δ(GGQTK) mutant (the eL42 protein whose GGQTK motif has been deleted), the single Q51E and K53Q mutants (eL42-Q51E and eL42-K53Q, respectively), as well as the double Q51A/K53A mutant (eL42-Q51A/K53A). RESULTS: Our results show that the monomethylated Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence of eL42 and the monomethylated GGQ motif of eRF1 represents the sites of interaction between these two proteins through hydrophobic contacts between methyl groups. We also demonstrate that the interactions between eL42 and tRNA or 28S rRNA are characterized by strong binding affinities (KD values in the nanomolar or picomolar range, respectively) which argue for specific interactions. Strong interactions between eL42 and tRNA are likely to be responsible for the decrease in the poly(U)-dependent poly(Phe) synthesis activity of human 80S or E. coli 70S ribosomes in the presence of added human recombinant eL42. It is proposed that the decrease of the activity of the ribosome is caused by the sequestration of the substrate Phe-tRNAPhe by the added eL42 protein. CONCLUSION: Interactions between the monomethylated Gln-51 and Lys-53 residues of the 49GGQTK53 motif of the human eL42 protein and the methylated GGQ motif of eRF1 are likely to play a functional role on translating human 80S ribosomes.

Exploring contacts of eRF1 with the 3'-terminus of the P site tRNA and mRNA stop signal in the human ribosome at various translation termination steps.

Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3'-terminus to investigate mutual arrangement of the main components of translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3'-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals.

EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance.

In this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elongator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E. coli or Frateuria sp. W-315, respectively, suggesting that antibiotic resistance of enacyloxin producing Frateuria sp. W-315 is not due neither to EF-Tu nor to other components of the translation machinery but to a still unknown mechanism. The EF-Tu gene, as PCR amplified from Frateuria W-315 genomic DNA and sequenced represented an ORF of 1191 nucleotides corresponding to 396 amino acids. This protein is larger than the product of tufA from E. coli by only two amino acid residues. Alignment of the amino acid sequence of EF-Tu from E. coli with those of Frateuria and Ralstonia solanacearum indicates on average 80% identical amino acid residues and 9.7% conservative replacements between EF-Tu Frateuria and EF-Tu E. coli, on one hand, and 97% identity and 1.7% conservative replacement between EF-Tu Frateuria and EF-Tu Ralstonia solanacearum, on the other hand. These strong primary structure similarities between EF-Tu from different origins are consistent with the fact that this factor is essential for the translation process in all kingdoms of life. Comparison of the effects of antibiotics on EF-Tu Frateuria and EF-Tu E. coli revealed that enacyloxin, kirromycin and pulvomycin exert a stronger stimulation of the GDP dissociation rate on EF-Tu Frateuria, while the effects of the antibiotics on the GDP association rate were comparable for the two EF-Tu species. Different mutants of EF-Tu E. coli were constructed with the help of site directed mutagenesis by changing one or several residues of EF-Tu E. coli by the corresponding residues of EF-Tu Frateuria. The single A45K substitution did not modify the intrinsic GTPase activity of EF-Tu E. coli. In contrast, a 2-3 fold stimulation of the intrinsic GTPase activity was observed with the single A42E, F46Y, Q48E and the double F46Y/Q48E substitution. Finally, up to a 7 fold stimulation was observed with the quadruple substitution (mutant A42E/A45K/F46Y/Q48E.

The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome.

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome.

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA(Asp) analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3'-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.

Lys53 of ribosomal protein L36AL and the CCA end of a tRNA at the P/E hybrid site are in close proximity on the human ribosome.

Previously we have shown that the CCA end of a P-tRNA can be crosslinked with the RPL36AL protein of the large subunit of mammalian ribosomes; it belongs to the L44e protein family present in all eukaryotic and archaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for a tRNA at the P/E hybrid site, as a tRNA in all other tRNA positions of pre-translocational ribosomes could not be crosslinked with a ribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E-tRNA, but could also connect the ultimate A of this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues, as well as glutaminyl residue 51), 4) Q51 is part of a conserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl-tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about 50 %, might indicate that protein RPL36AL is a preferential target for regulation.

DNA-adducts in subjects exposed to urban air pollution by benzene and polycyclic aromatic hydrocarbons (PAHs) in Cotonou, Benin.

Air pollution effect on humans represents a major public health problem. Exposure to genotoxic compounds in the ambient air is evaluated using different biomarkers. In the present study we assessed DNA-adducts levels in apparently healthy people living and working in the city of Cotonou (Benin) in which exposure to air pollutants such as benzene and polycyclic aromatic hydrocarbons (PAHs) mainly benzo(a)pyrene has been evidenced. Rural inhabitants were enrolled as control group. Taxi-motorbike drivers, street food vendors, and gasoline salesmen were recruited in Cotonou whereas suburban residents were recruited in Godomey, 12 km from Cotonou. We found that taxi-motorbike drivers, roadside residents, street vendors, taxi-motor-bike drivers and gasoline sellers had significantly higher levels of DNA-adducts than suburban and village inhabitants (P < 0.001; post hoc, LSD). Means values were 24.6 ± 6.4, 23.78 ± 6.9, 34.7 ± 9.8, and 37.2 ± 8.1 in the exposed groups versus 2.1 ± 0.6 and 3.1 ± 0.8 adducts/10(8) nucleotides, in the two control groups, respectively. We did not find any significant difference within the high exposure groups and inside low exposure subgroups (namely suburban residents and villagers) because the mean individual exposure values to both PAHs and benzene were similar among subjects exposed in the city of Cotonou and those in suburban and village areas. However, there is significant interindividual variations in adducts levels that may reflect variation of genetic susceptibility factors. Ranges of adduct level/10(8) nucleotides were: 1-69, 1-76, 3-169, 4-124, 0-9, 0-8 adducts/10(8) for taxi-motorbike drivers, roadside residents, street vendors, gasoline sellers, suburban and village inhabitants, respectively. Our study demonstrated a clear-cut elevated level of DNA adducts in city residents than in none exposed people (or very low exposure levels people) and designate these city residents groups as people at risks for the chronic diseases possibly caused by benzene and PAHs.

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA.

Periodate-oxidized tRNA (tRNAox), the 2',3'-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3'-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA(Asp)ox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA(Asp) competed with tRNA(Asp)ox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.

Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry.

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNA(Ile), the 2',3'-dialdehyde derivative of tRNA(Ile) was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNA(Ile) aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNA(Ile)ox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNA(Ile) and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence (601)KMSKS(605). Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [(32)P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.

Arrangement of 3'-terminus of tRNA on the human ribosome as revealed from cross-linking data.

This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3'-end of tRNA in the 80S ribosomal A and P sites using a tRNA(Asp) analogue that bears a 4-thiouridine (s(4)U) attached to the 3'-terminal adenosine. It was shown that an additional nucleotide s(4)U77 on the 3'-end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302-4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides cross-linked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s(4)U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3'-terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.

The search for traces of life: the protective effect of salt on biological macromolecules.

Trapping malate dehydrogenase from the extremely halophilic archaeon Haloarcula marismortui in "dry" salt crystals protects the enzyme against thermal denaturation. Similar protection was not observed for the homologous mesophilic enzyme. In the case of transfer RNA molecules, high salt concentration plays a protective role against thermal degradation allowing activity to be recovered. The results are discussed in the context of exploring the fate of cell-free biological macromolecules in the environment and that of orienting the search for traces of life in planetary exploration.