Jump to main content. Jump to site search. You do not have JavaScript enabled. Please enable JavaScript to access the full features of the site or access our non-JavaScript page. Issue 53, From the journal: RSC Advances. This article is Open Access. Please wait while we load your content Something went wrong. Try again? Cited by. The mechanism of this exclusive use of L-amino acids is not yet fully understood.

This is especially notable in bacteria whose cytosol contains about a dozen different D-amino acids that are used as a carbon source, signaling molecules, or building blocks for peptidoglycan cell wall synthesis 1 , 2.

In some bacteria, D-amino acids are present in millimolar concentrations, sometimes with the levels of D-isomers exceeding those of their L-isomers as in the case of D-alanine and D-glutamate 2 , 3. In eukaryotes, nano- to micromolar concentrations of D-amino acids are typically present in animals, plants, and fungi 4 , 5. Thus, organisms, from bacteria to higher eukaryotes, utilize only the L-amino acids for protein synthesis despite the presence of D-amino acids in cell cytosol.

The exclusion of D-amino acids from the ribosome-dependent protein synthesis is achieved through the cooperation of at least four independent mechanisms. First, the aminoacyl-tRNA-synthetases, which select amino acids for protein synthesis, react markedly slower with D-amino acids than with L-amino acids.

This enzyme is conserved across the three domains of life and prevents accumulation and toxicities of D-aminoacyl-tRNAs 9. Finally, in vitro studies showed that if a D-aminoacyl-tRNA binds the ribosomal A site, it reacts with a P-site substrate at about three orders of magnitude slower rate compared to L-aminoacyl-tRNAs, illustrating that D-amino acids markedly reduce the rate of the peptide-bond formation If a D-amino acid is incorporated into a nascent peptide and translocated to the P site, it might cause translation arrest, suggesting that D-amino acids also interfere with the passage of the nascent peptide through the ribosomal exit tunnel Thus, cells have intricate fidelity control systems that favor preferential usage of the L-isomers over the D-amino acids at every stage of protein synthesis.

In the past years, the interest to the D-amino acid recognition by the ribosome has been revitalized due to progress in the genetic code expansion of living cells 12— However, all of the amino acids that have been successfully incorporated in vivo comprise only the L-isomers, while genetic encoding of D-amino acids remains a challenge. Ribosomal synthesis of proteins with D-amino acids is desired, because site-specific replacement of L-amino acid residues with their D-isomers renders corresponding peptides protease-resistant, as it was shown for hormones and other pharmacologically active polypeptides 16— Also, D-amino acids are present in natural proteins introduced via post-translational isomerization , such as bacterial lantibiotics, opioid peptides from frogs, and conotoxins Therefore, the ability to perform ribosomal synthesis of D-amino acid-containing proteins is required to enable the large-scale and cost-effective production of pharmacologically active proteins and peptides.

Over the past years, messenger RNA-dependent synthesis of D-amino acid-containing proteins became possible in vitro via engineering of different translation machinery components. For example, protein engineering allowed the creation of aminoacyl-tRNA synthetases that selectively use D-isomer of tyrosine 25 , Also, development of engineered catalytic RNAs, flexizymes, made possible production of D-aminoacyl-tRNAs for use in cell-free protein translation systems Optimization of in vitro translation systems allowed synthesis of detectable amounts of peptides containing up to 10 consecutive D-amino acids 28— However, these ribosome mutants were less accurate and highly toxic in Escherichia coli preventing their use in vivo The rational engineering of ribosomes to enable efficient usage of D-amino acids is currently limited due to the lack of a structural basis for the poor reactivity of D-amino acids in the peptide bond formation reaction.

Our structure reveals that the D-aminoacyl-tRNA analog binds the ribosome in a similar fashion as L-aminoacyl-tRNAs with the CCA-end binding the ribosomal A site in a canonical way and with the D-amino acid side chain accommodated by the ribosomal side chain-binding pocket. Thus, our study reconciles the observed poor reactivity of D-amino acids in ribosomal protein synthesis. The reported structure provides an essential framework for the future rational design of the PTC and its surroundings to improve the usage of D-amino acids by the ribosome.

The ACCA-D-Phe conjugate was produced as outlined in Figure 1A and as described below similar to the synthesis schemes previously reported in references 34— N - 9-Fluorenyl methoxycarbonyl-D-phenylalanine compound 2. D-Phenylalanine 1 0. At this point, 9-fluorenylmethoxycarbonyl chloride 0.

The ice bath was removed and the reaction mixture was stirred for 7 h at room temperature and afterwards the reaction mixture was acidified with concentrated HCl to pH 2. The resulting solution was extracted with dichloromethane ml , the organic phase was washed twice with H 2 O 50 ml and dried over Na 2 SO 4.

Yield: 1. Fmoc-protected D-phenylalanine 2 75 mg, 0. To a solution of compound 4 80 mg, 0. The mixture was stirred for one hour followed by evaporation of the solvents.

The crude ester 53 mg, 0. For capping of unreacted amino groups, the beads were treated with a mixture of solution Cap A 0. The suspension was filtrated again; the beads were washed with acetonitrile, methanol, and CH 2 Cl 2 and dried under vacuum.

The deprotection and cleavage of the conjugate from the solid support proceeded in three steps. Fmoc deprotection. Acyl deprotection and cleavage from the solid support. After 4-hour shaking at room temperature, the supernatant was filtered and evaporated to dryness. By eluating with H 2 O, the conjugate-containing fractions were collected and evaporated to dryness, and the residue was dissolved in H 2 O 1 ml.

Conjugate-containing fractions were evaporated to dryness and dissolved in H 2 O 1 ml. The quality of the purified conjugate was analyzed by analytical anion-exchange chromatography Figure 1B. Yields were determined by UV photometric analysis of conjugate solutions. Chemical synthesis of a short hydrolysis-resistant D-phenylalanyl-tRNA analog. A Synthetic route.

Letters indicate specific reaction conditions as follows: a 1. Initial crystalline needles were obtained by screening around previously published ribosome crystallization conditions 38— A complete dataset for each ribosome complex was collected using 0. The raw data were integrated and scaled using the XDS software package The search model was generated from the previously published structure of the T. The initial molecular replacement solutions were refined by rigid body refinement with the ribosome split into multiple domains, followed by 10 cycles of positional and individual B-factor refinement using PHENIX Non-crystallographic symmetry restraints were applied to 4 domains of the 30S ribosomal subunit head, body, spur, helix 44 , and four domains of the 50S subunit body, L1-stalk, Lstalk, C-terminus of the L9 protein.

The statistics of data collection and refinement are compiled in Table 1. All figures showing atomic models were generated using PyMol software www. The electron density maps allow to unambiguously position L- and D-amino acid side chains bound to the ribosomal active site.

The amino acid moieties of L-methyl-tyrosine and D-phenylalanine are highlighted in blue and red, respectively. The refined model of each compound is displayed in its respective electron density map before the refinement green mesh. Nitrogens are colored blue; oxygens are red; phosphorus atoms are orange. Each of the difference electron density maps is contoured at 2.

E, F Comparison of the current structures with the previously reported structures of the A-site-bound short and full-length tRNA substrates. Note that differences between the compared structures of the A-site substrates are within experimental error. To provide structural insights into the poor reactivity of the D-aminoacyl-tRNAs in the peptide bond formation we determined the crystal structure of T.

Using either of these compounds as an A-site substrate, we determined their crystal structures in complex with the T. Although the P-site tRNA in both of our complexes was represented by the deacylated tRNA i fMet , which is not strictly physiological, previous studies have shown that aminoacylation status of the P-site tRNA does not affect conformation of the amino acid attached to the A-site tRNA substrate 37 , 47— Therefore, it is reasonable to assume that the conformation and interactions of the A-site substrates in our structures are identical to those seen in physiologically more relevant complexes of the ribosome.

Both crystal structures were determined at 3. To build the structural models of the A-site substrates, we used the best-fit placement of ACCA-D-Phe and CC-Pmn molecules into the electron density maps and subsequent crystallographic refinement Materials and Methods. Because the resolution of our datasets did not allow direct visualization of the individual chemical groups, the accurate model building was aided by the chemical restraints.

The only reason why we determined it again is to validate the accuracy of our structural models determined at 3. We next asked if our maps provide a sufficient level of detail to gain mechanistic insights into ribosome stereoselectivity. For this purpose, we compared our 3. Our comparison revealed no significant differences in the location and orientation of the CC-Pmn molecule on the 70S ribosome between the new and the previous structures Figure 2E.

In all analyzed structures, the L-mTyr was tightly fit into the A-site pocket due to shape complementarity between the ribosomal A-site and the amino acid backbone. This similarity of L-mTyr conformation in different crystal structures illustrated that, despite limited detail, our 3.

Our electron density maps revealed that, in both crystal structures, the CCA-ends of aminoacyl-tRNA analogs establish canonical contacts with the A-loop Figure 3B , illustrating that the presence of D amino acid residue does not impede recognition of the CCA-end by the ribosome Figure 2E , F.

D-aminoacyl-tRNA analog establishes canonical A-loop interactions. Note that these A-loop interactions play a key functional role in accommodation and proper positioning of the substrate in the A site of the ribosome. We next asked how D-amino acid binding to the A site is compared to that of L-amino acids.

The electron density maps revealed the backbones of both L- and D- amino acids, as well as the entire side chain of the L-methyl-tyrosine Figures 2C and 4C , D. Thus, our data indicate that not only the CCA-end of the D-aminoacyl-tRNA analog forms canonical interactions with the ribosome but also the D-amino acid side chain binds the A-site cleft in a fashion similar to that of the L-amino acids side chains before the peptide bond formation takes place.

Side chains of both L- and D-amino acids occupy the A-site cleft of the ribosome. The E. The ability of this group to attack the P-site substrate from this remote location is expected to be reduced due to the non-optimal geometry curved red arrow.

Note that the superimposed tRNAs structures are nearly identical even though one is determined at 3. Of course, at the 3. What is crucial here is the fact that we observe electron density for the part of the D-Phe side chain. Reactive conformation of the D-amino acid is likely prevented by the conserved rRNA residues in the peptidyl-transferase center.

Relative locations of the 23S rRNA residues A and C forming the A-site cleft light blue spheres and the residues G, A, U and A, whose mutations improve utilization of the D-amino acids by the ribosome blue. Note that residues G and A blue spheres are located near the A-site cleft. Mutations of these purine residues to smaller pyrimidines might lead to either an increased size of the A-site cleft or increased flexibility of the adjacent residues forming the A-site cleft.

To answer this question, we explored the range of sterically allowed conformations of the D-phenylalanine in the ribosomal A site by using in silico modeling. This structure provides mechanistic insights into the poor reactivity of D-amino acids in the peptide bond formation and illustrates one of the mechanisms that allow cells to prevent co-translational incorporation of D-amino acids into natural proteins.

A-site cleft binds the side chains of incoming amino acids and plays a critical role in the positioning of the incoming amino acids in the PTC. Such orientation was suggested to help physically exclude amino acid side chain from the catalytic center of the ribosome and, thereby, prevent potential steric clashes and non-desired chemical reactivities of amino acid side chains in the peptide bond formation. Our observation that the D-amino acid side chain accommodates into the A-site cleft suggests that this conserved component of the ribosomal catalytic center might also be involved in the stereospecificity control of protein synthesis.

Previous studies suggested two alternative models of how ribosome can discriminate between the two possible chiralities of the incoming amino acids and reject D-amino acids from the use in protein synthesis. One model, based on the molecular modeling attempts using pioneering structures of archaeon Haloarcula marismortui , assigned the critical role in rejecting D-amino acids to the nucleotide U in the 23S rRNA 52 , In another model, based on structural analysis of the pre-attack state of the peptide bond formation reaction, the critical role in discriminating amino acid chirality has been assigned to the nucleotide U The main difference between these two models stemmed from the lack of knowledge about the orientation of the D-amino acid in the ribosomal A site, particularly regarding the orientation of the side chain.

Our current structure, illustrating how the D-amino acid side chain binds the A-site cleft of the ribosome, is consistent with the model in which the key discriminatory role is played by the U residue Figure 5B. Also, our study might explain previous findings that the use of D-amino acids by the ribosome can be improved through the mutagenesis of the 23S rRNA 32 , Mutations of the purine nucleotides G and A to smaller pyrimidines should increase the size of the A-site cleft, thereby allowing the D-amino acids to adopt more reactive conformations without clashing of their side chains with the residues of the PTC.

Over the past two decades, ribosome engineering produced an array of ribosome variants for applications in basic research and biotechnology Ribosomes with mutated anti-Shine-Dalgarno sequence 56 and with tethered ribosomal subunits 57 , 58 were constructed to allow the presence of two independent translation systems in a single cell. Hybrids between bacterial and eukaryotic ribosomes were constructed to explore principles of antibiotic specificity 59 or use bacterial ribosomes in eukaryote-derived in vitro translation systems Also, engineered ribosomes were produced to decode quadruplet codons 61 or recognize artificial tRNAs By showing D-amino acid residue in the ribosomal catalytic center, our structure provides the basis for the rational design of the amino acid binding pocket to improve ribosome compatibility with non-canonical substrates.

In this regard, it is important to note that ribosomes with altered A-site cleft have been previously observed in nature. However, they did find that this mutated version of subtilisin still acted as a catalyst, and would break peptide bonds times faster than would occur with no catalyst. Thus even earlier forms of these enzymes, those that existed before the catalytic triad mechanism had evolved into their structure, would still be valuable to the organisms in which they had originally evolved.

There are other large groups of proteases that share common mechanisms, but do not use an active serine. For example, there are cysteine peptidases, such as papain from papaya, in which the sulfur in cysteine acts much like the oxygen on serine in serine proteases.

There are also aspartic acid peptidases, which include HIV protease, an important target for drug development. There are a wide range of metalloproteases that use the interaction of a specific metal ion to help the reaction Figure 3. And there are proteases for which scientists have not yet discovered the mechanism of action. Figure 3: Protease mechanisms Polypeptides can be cleaved either chemically or enzymatically.

Enzymes that catalyse the hydrolytic cleavage of peptide bonds are called proteases. Proteases fall into four main mechanistic classes: serine, cysteine, aspartyl and metalloproteases. In the active sites of serine and cysteine proteases, the eponymous residue is usually paired with a proton-withdrawing group to promote nucleophilic attack on the peptide bond. Aspartyl proteases and metalloproteases activate a water molecule to serve as the nucleophile, rather than using a functional group of the enzyme itself.

However, the overall process of peptide bond scission is essentially the same for all protease classes. Soluble serine proteases a ; cysteine proteases b ; aspartyl proteases c ; and metalloproteases d.

How intramembrane proteases bury hydrolytic reactions in the membrane. No single mechanism can explain the wealth of chemical reactions carried out by biological enzymes. They can transfer electrons and protons, assemble small molecules into large molecules and break them back into smaller pieces, accurately copy DNA, and repair DNA when it is damaged. Above all these specific functions, there is indeed one commonality among all enzymes; they carry out reactions identical to the types of reactions carried out by organic chemists.

Over the course of evolution, enzymes have become astounding catalysts. In the words of one prominent scientist, enzymes are not different, they are just better Knowles Scientists have combined data from chemical labeling studies, X-ray crystallography, and planned mutations to gain an understanding of how serine proteases can accelerate the rate of peptide bond hydrolysis.

They have been able to identify specific amino acids involved in the catalytic steps used by these enzymes. They have also been able to recreate enzymes that may have been intermediate versions of these enzymes during evolution. However, we are still unable to fully predict how small amino acid changes can alter the structure and function of proteins. Scientists are continuing to discover new members of this diverse protein family, which is vital to the life of all organisms.

Allen, K. Nature Chemical Biology 6, June doi: Blow, D. Role of a buried acid group in the mechanism of action of chymotrypsin. Brenner, S. The molecular evolution of genes and proteins: a tale of two serines. Carter, P. Dissecting the catalytic triad of a serine protease. Craik, C. Redesigning trypsin: alteration of substrate specificity. Science , — Delbaere, L. Tertiary structural differences between microbial serine proteases and pancreatic serine enzymes.

Dixon, G. Biochimica et Biophysica Acta 20, — Erez, E. Hartly, B. The reaction of p-nitrophenyl esters with chymotrypsin and insulin. Biochemical Journal 56, — Kahne, D. Hydrolysis of a peptide bond in neutral water. Journal of the American Chemical Association , — Knowles, J. Enzyme catalysis: not different, just better.

Puente, X. Rawlings, N. Rose, T. Serine Protease Home Page. Schoellmann, G. Direct evidence for the presence of histidine in the active center of chymotrypsin. Biochemistry 2, — Stroud, R. A family of protein cutting enzymes. Scientific American , 74—88 Ribosomes, Transcription, and Translation. Gene Expression. Protein Structure. Protein Function. Analyzing Regulatory Networks in Bacteria.

Aging and Cell Division. Calreticulin: a Multifaceted Protein. Enzyme Catalysis: The Serine Proteases. Germ Cells and Epigenetics. MicroRNAs in Arabidopsis. Protein Misfolding and Degenerative Diseases. Systems Biology of the Cell. Neitzel, Ph. Citation: Neitzel, J. Nature Education 3 9 How do enzymes cause tremendous increases in reaction rates?

Learn how a group of enzymes called the serine proteases work. Aa Aa Aa.