Ribosomal crystallography: peptide bond formation, chaperone assistance and antibiotics activity

The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3' ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A- to P-site passage of the tRNA 3' end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by gene-fusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent chain trafficking into the hydrophobic space formed by the first bacterial chaperone, the trigger factor. Many antibiotics target ribosomes. Although the ribosome is highly conserved, subtle sequence and/or conformational variations enable drug selectivity, thus facilitating clinical usage. Comparisons of high-resolution structures of complexes of antibiotics bound to ribosomes from eubacteria resembling pathogens, to an archaeon that shares properties with eukaryotes and to its mutant that allows antibiotics binding, demonstrated the unambiguous difference between mere binding and therapeutical effectiveness. The observed variability in antibiotics inhibitory modes, accompanied by the elucidation of the structural basis to antibiotics mechanism justifies expectations for structural based improved properties of existing compounds as well as for the development of novel drugs.     

Eryngin, a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii

An antifungal peptide with a molecular mass of 10k Da was isolated from fruiting bodies of the mushroom Pleurotus eryngii. The peptide, designated as eryngin, inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola. It was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and S-Sepharose. Its N-terminal sequence demonstrated some similarity to the antifungal protein from the mushroom Lyophyllum shimeiji and little resemblance to thaumatin and thaumatin-like proteins.     

Ribosomal tolerance and peptide bond formation

In the ribosome, the decoding and peptide bond formation sites are composed entirely of ribosomal RNA, thus confirming that the ribosome is a ribozyme. Precise alignment of the aminoacylated and peptidyl tRNA 3'-ends, which is the major enzymatic contribution of the ribosome, is dominated by remote interactions of the tRNA double helical acceptor stem with the distant rims of the peptidyl transferase center. An elaborate architecture and a sizable symmetry-related region within the otherwise asymmetric ribosome guide the A --> P passage of the tRNA 3'-end by a spiral rotatory motion, and ensures its outcome: stereochemistry suitable for peptide bond formation and geometry facilitating the entrance of newly formed proteins into their exit tunnel.     

Optimization of enzyme-mediated peptide bond formation

Enzyme-catalyzed peptide bond formation requires thorough examination and optimization of each coupling step. In order to identify factors influencing the selectivity between aminolysis and hydrolysis, a systematic study was carried out for the kinetically controlled peptide synthesis. The reaction temperature, the type of C-terminal protecting group, and different organic cosolvents showed little influence on the selectivity. The enzyme, excess nucleophile, pH, N-terminal protecting group, and ionic strength of the solution were identified as major factors controlling the selectivity and, therefore, the yield of the dipeptide synthesis. Under optimized conditions, the selectivity of the chymotrypsin-catalyzed synthesis of PheSer could be increased from 35 to 100%.     

Gene cloning and biochemical characterization of eryngase,a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH₂ as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH₂ by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH₂. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH₂ substrate.     

Biology of Pleurotus eryngii and role in biotechnological processes: a review

Pleurotus eryngii is considered a complex species owing to a perplexed structure within species and a wide geographical distribution. Due to its remarkable flavor, high nutritional value, and numerous medicinal features, P. eryngii is commercially cultivated on various raw plant materials. Its efficacy in using nutrients from lignocellulose residues is based on possession of a potent ligninolytic enzyme system, constituted of laccase, Mn-oxidizing peroxidases, and aryl-alcohol oxidase, which successfully degrade different aromatic compounds. Similarly, due to the ability of these enzymes, P. eryngii plays a very important role in many biotechnological processes, such as food production (edible basidiomata), biotransformation of raw plant materials to feed, biopulping and biobleaching of paper pulp, as well as bioremediation of soil and industrial waters.     

Ribosomal crystallography: peptide bond formation and its inhibition

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.     

Catechol as a nucleophilic catalyst of peptide bond formation.

The aminolysis of a mildly activated aminoacid ester, benzyloxycarbonyl-L-phenylalanine cyanomethyl ester, by glycine esters in the presence of catechol has been studied as a model of catalysis by RNA cis-vicinal-diol systems in protein biosynthesis. Catechol accelerated the aminolysis, especially in the presence of bases, probably by nucleophilic catalysis.     

Isolation and characterization of a sporeless mutant in Pleurotus eryngii

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.     

Mutation affecting peptide bond formation in nikkomycin biosynthesis

Nikkomycin, a nucleoside-peptide analog of UDP-N-acetylglucosamine, is a potent chitin synthase inhibitor produced by the bacterium Streptomyces tendae. The HPLC profile of fermentation products in culture broths of a non-producing mutant, Nik 15, was compared with nikkomycin standards. Nikkomycin C and D, the glycone and aglycone moieties, respectively, of nikkomycin Z accumulated. This indicates the mutation affects the capacity to form a peptide bond between nikkomycin C and D, which is here proposed to be the terminal step in the synthesis of the biologically active nikkomycin Z. This is also the first documented case of a mutation affecting a specific step in nikkomycin biosynthesis.     

Mechanism of peptide bond formation on the ribosome

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.     

Mechanism of peptide bond formation on the ribosome--controversions

During past five years there have been published many experimental data concerning structure and function of the ribosome. With the presentation of atomic structures we obtained a new data about composition of peptidyl transferase center. It is now obvious that PTC is composed entirely of rRNA. It is also known that the proper substrate alignment is the major factor for ribosome's catalytic activity. However, more detailed mechanism of peptide bond formation on the ribosome still remains unclear. Several issues remain unsolved. For example, are there any chemical components coming from ribosome itself, that enhance the rate of the reaction? Do intact ribosomes perform peptidyltransfer in the same way as the isolated ribosomal subunits that have been the source of most of the data? In this article we present different opinions and controversions around peptide bond formation on the ribosome.     

Protein evolution: intrinsic preferences in peptide bond formation: a computational and experimental analysis

Two possibilities exist for the evolution of individual enzymes/proteins from a milieu of amino acids, one based on preference and selectivity and the other on the basis of random events. Logic is overwhelmingly in favour of the former. By protein data base analysis and experiments, we have provided data to show the manifestation of two types of preferences, namely, the choice of the neighbour and its acceptance from the amino end (left) or the carboxyl end (right). The study tends to show that if the 20 proteinous amino acids were made to combine in water, the resulting profile would be nonrandom. Such selectivity could be a factor in protein evolution. Dedicated to the memory of Darshan Ranganathan.     

Energetics of peptide bond formation at elevated temperatures

Summary The free energies of formation of the peptide bond between carbobenzoxy-glycine and L-phenylalanine amide in aqueous solution at temperatures up to 60°C were calculated from experimentally determined equilibrium constants. The reaction was catalyzed by a thermophylic enzyme. The thermodynamic energy barrier to peptide bond formation was found to decrease with increasing temperature: the standard free energy of peptide bond formation did appear to become negative in the region of 60°C. The possible significance of these results for peptide bond formation under prebiotic conditions is discussed.     

Strophasterols E and F: Rearranged ergostane-type sterols from Pleurotus eryngii

Strophasterols E (1) and F (2) were isolated from the fruiting bodies of Pleurotus eryngii, together with four new ergostane-type sterols (3–6). Single-crystal X-ray diffraction analysis performed on the tris-p-bromobenzoate derivatives of compounds 1 and 2 allowed these two compounds to be identified as the structurally rare (22S,23R)- and (22S,23S)-5α,6α-epoxy-3β,7β,23-trihydroxy-15(14 → 22)-abeo-ergost-8-en-14-one, respectively. The inhibitory effects on nitric oxide production of the six new steroids thus isolated from the fruiting bodies of P. eryngii were also evaluated.     

Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

Summary The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic protoenzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.