Construction of modified ribosomes for incorporation of D-amino acids into proteins

While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.     

Simultaneous determination of D-amino acids by the coupling method of D-amino acid oxidase with high-performance liquid chromatography

An enzymatic assay system of D-amino acids was established using the D-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the D-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of L-amino acids, which are generally present at much higher concentrations than D-amino acids in biological samples. With this method, D-Glu, D-Asn, D-Gln, D-Ala, D-Val, D-Leu, D-Phe, and D-Ile can be quantified in the order of micromolar, and other D-amino acids except D-Asp can be assayed within a sensitivity range of 50-100 μM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of D-Ser obtained using this enzymatic method (223 μM) was in good agreement with that obtained using the conventional HPLC method (198 μM). The enzymatic method also demonstrated that the human urine contained 5.45 μM of d-Ala and 0.91 μM of D-Asn. Both D-amino acids were difficult to be identified using the conventional method, because the large signals from L-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem.     

D-Amino acids and D-Tyr-tRNA(Tyr) deacylase: stereospecificity of the translation machine revisited

Until 30 years ago, it had been considered that D-amino acids were excluded from living systems except for D-amino acids in the cell wall of microorganisms. However, D-amino acids, in the form of free amino acids, peptides and proteins, were recently detected in various living organisms from bacteria to mammals. The extensive distribution of bio-functional D-amino acids challenges the current concept of protein synthesis: more attention should be paid to the stereospecificity of the translation machine. Besides aminoacyl-tRNA synthetases, elongation factor Tu and some other mechanisms, D-Tyr-tRNA(Tyr) deacylases provide a novel checkpoint since they specifically recycle misaminoacylated D-Tyr-tRNA(Tyr) and some other D-aminoacyl-tRNAs. Their unique structure represents a new class of tRNA-dependent hydrolase. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origin.     

Cloning and functional characterization of Arabidopsis thaliana D-amino acid aminotransferase--D-aspartate behavior during germination

The understanding of D-amino acid metabolism in higher plants lags far behind that in mammals, for which the biological functions of these unique amino acids have already been elucidated. In this article, we report on the biochemical behavior of D-amino acids (particularly D-Asp) and relevant metabolic enzymes in Arabidopsis thaliana. During germination and growth of the plant, a transient increase in D-Asp levels was observed, suggesting that D-Asp is synthesized in the plant. Administration of D-Asp suppressed growth, although the inhibitory mechanism responsible for this remains to be clarified. Exogenous D-Asp was efficiently incorporated and metabolized, and was converted to other D-amino acids (D-Glu and D-Ala). We then studied the related metabolic enzymes, and consequently cloned and characterized A. thaliana D-amino acid aminotransferase, which is presumably involved in the metabolism of D-Asp in the plant by catalyzing transamination between D-amino acids. This is the first report of cDNA cloning and functional characterization of a D-amino acid aminotransferase in eukaryotes. The results presented here provide important information for understanding the significance of D-amino acids in the metabolism of higher plants.     

Nonribosomal biosynthesis of peptide antibiotics

Peptide antibiotics are known to contain non-protein amino acids, D-amino acids, hydroxy acids, and other unusual constituents. In addition they may be modified by N-methylation and cyclization reactions. Their biosynthetic origin has been connected in many cases to an enzymatic system referred to as the 'thiotemplate multienzymic mechanism'. This mechanism includes the activation of the constituent residues as adenylates on the enzymic template, the acylation of specific template thiol groups, epimerization or N-methylation at this thioester stage, and polymerization in the sequence directed by the multienzymic structure with the aid of 4'-phosphopantetheine as a cofactor, including possible cyclization or terminal modification reactions. The reaction sequences leading to gramicidin S, tyrocidine, cyclosporine, bacitracin, polymyxin, actinomycin, enniatin, beauvericin, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and linear gramicidin are discussed. The structures of the multienzymes, their genetic organization, the biological functions of these peptides and results on related systems are discussed.     

D-Amino Acids in Living Higher Organisms

The homochirality of biological amino acids (L-amino acids) andof the RNA/DNA backbone (D-ribose) might have become establishedbefore the origin of life. It has been considered that D-aminoacids and L-sugars were eliminated on the primitive Earth.Therefore, the presence and function of D-amino acids in livingorganisms have not been studied except for D-amino acids in thecell walls of microorganisms. However, D-amino acids wererecently found in various living higher organisms in the form offree amino acids, peptides, and proteins. Free D-aspartate andD-serine are present and may have important physiologicalfunctions in mammals. D-amino acids in peptides are well knownas opioid peptides and neuropeptides. In protein, D-aspartateresidues increase during aging. This review deals with recentadvances in the study of D-amino acids in higher organisms.     

Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D-amino acids were substituted for the L-amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L-peptide) rich in five arginine residues and consisting of an 11-amino acid peptide (residues 32-42) of human granulysin was synthesized. Subsequently, the L-amino acids of the 11-amino acid peptide were replaced partially (D-peptide) or wholly (AD-peptide) with D-amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D-amino acid substitutions were examined. Peptides with D-amino acid substitutions were found to lyse bacteria as efficiently as their all-L-amino acid parent, L-peptide. In addition, the peptide composed of L-amino acids was susceptible to trypsin, whereas peptides containing D-amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L-amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS-incubated peptide. Thus, D-amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D-amino acids are potential candidates for clinical therapeutic use.     

Mirrors in the PDB: left-handed α-turns guide design with D-amino acids

Background  

Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids.     

D-Amino acids in chronic renal failure and the effects of dialysis and urinary losses

Summary Total D-amino acids were measured in plasma for 20 non-dialysed patients (creatinine clearance < 12 ml/minute), 20 on CAPD, 20 on haemodialysis and 20 normals. Plasma D-tyrosine and D-phenylalanine were measured in 8 of each group by HPLC. Total D-amino acids, D-tyrosine and D-phenylalanine were significantly greater for patients than normals. D-amino acids and D-tyrosine correlated with creatinine and were decreased during HD. During dialysis, the mean losses for D-tyrosine and D-phenylalanine were similar, about 0.2 mg/CAPD exchange and 3 mg/4 hour haemodialysis (i.e. 2% of the total amino acid, as in plasma). Clearance was unaffected by stereochemical configuration. Urinary losses/24 hour in the non-dialysed patients were 0.35 mg D-tyrosine and 0.25 mg D-phenylalanine. Clearance for D-phenylalanine was greater than for the L-enantiomer. Increases in D-amino acids in renal failure are probably due to depletion of D-amino acid oxidase, but may be enhanced by a D-amino acid rich diet, peptide antibiotics and D-amino acid oxidase inhibiting drugs and metabolites. Possible toxic effects need further investigation.     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

Esterification of Escherichia coli tRNAs with D-histidine and D-lysine by aminoacyl-tRNA synthetases

It is generally believed that only L-amino acids are acceptable in protein synthesis, though some D-amino acids, including D-tyrosine, D-aspartate, and D-tryptophan are known to be bound enzymatically to tRNAs. In this report, we newly show that D-histidine and D-lysine are also able to be the substrates of respective Escherichia coli aminoacyl-tRNA synthetases.     

Microdetermination of serum D-amino acids

A method for the quantitative determination of serum D-amino acids in the range 0.5-20 nmol is described. In the method alpha-keto acids, derived from D-amino acids by D-amino acid oxidase, are measured as hydrazones. The method is unresponsive to the presence of a large excess of L-amino acids. It allows a fast assay in a small amount of specimen (0.1 ml), with good reproducibility and accuracy.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

Spatiotemporal localization of D-amino acid oxidase and D-aspartate oxidases during development in Caenorhabditis elegans

Recent investigations have shown that a variety of D-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. D-Amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are degradative enzymes stereospecific for D-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of D-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several D- and L-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and D-amino acids in multicellular organisms.     

Occurrence of D-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum

The contents of D-enantiomers of serine, alanine, proline, glutamate (glutamine) and aspartate (asparagine) were examined in the membrane fractions, soluble proteins and free amino acids from some species of archaea, Pyrobaculum islandicum, Methanosarcina barkeri and Halobacterium salinarium. Around 2% (D/D+L) of D-aspartate was found in the membrane fractions. In the soluble proteins, the D-amino acid content was higher in P. islandicum than that in the other archaeal cells: the concentrations in P. islandicum were 3 and 4% for D-serine and D-aspartate, respectively. High concentrations of free D-amino acids were found in P. islandicum and H. salinarium; the concentrations of D-serine (12-13%), D-aspartate (4-7%) and D-proline (3-4%) were higher than those of D-alanine and D-glutamate. This result showed a resemblance between these archaea and not bacterial, but eukaryotic cells. The presence of D-amino acids was confirmed by their digestion with D-amino acid oxidase and D-aspartate oxidase. The occurrence of D-amino acids was also confirmed by the presence of activities catalyzing catabolism of D-amino acids in the P. islandicum homogenate, as measured by 2-oxo acid formation. The catalytic activities oxidizing D-alanine, D-aspartate and D-serine at 90 degrees C were considerably high. Under anaerobic conditions, dehydrogenase activities of the homogenate were 69, 84 and 30% of the above oxidase activities toward D-alanine, D-aspartate and D-serine, respectively. Comparable or higher dehydrogenase activities were also detected with these D-amino acids as substrate by the reduction of 2, 6-dichlorophenolindophenol. No D-amino acid oxidase activity was detected in the homogenates of M. barkeri and H. salinarium.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

D-Amino acids in mammals and their diagnostic value

Substantial amounts of D-amino acids are present in mammalian tissues; their function, origin and relationship between pathophysiological processes have been of great interest over the last two decades. In the present article, analytical methods including chromatographic, electrophoretic and enzymatic methods to determine D-amino acids in mammalian tissues are reviewed, and the distribution of these D-amino acids in mammals is discussed. An overview of the function, origin and relationship between the amino acids and pathophysiological processes is also given.     

Human D-Tyr-tRNA(Tyr) deacylase contributes to the resistance of the cell to D-amino acids

DTD (D-Tyr-tRNA(Tyr) deacylase) is known to be able to deacylate D-aminoacyl-tRNAs into free D-amino acids and tRNAs and therefore contributes to cellular resistance against D-amino acids in Escherichia coli and yeast. We have found that h-DTD (human DTD) is enriched in the nuclear envelope region of mammalian cells. Treatment of HeLa cells with D-Tyr resulted in nuclear accumulation of tRNA(Tyr). D-Tyr treatment and h-DTD silencing caused tRNA(Tyr) downregulation. Furthermore, inhibition of protein synthesis by D-Tyr treatment and h-DTD silencing were also observed. D-Tyr, D-Asp and D-Ser treatment inhibited mammalian cell viability in a dose-dependent manner; overexpression of h-DTD decreased the inhibition rate, while h-DTD-silenced cells became more sensitive to the D-amino acid treatment. Our results suggest that h-DTD may play an important role in cellular resistance against D-amino acids by deacylating D-aminoacyl tRNAs at the nuclear pore. We have also found that m-DTD (mouse DTD) is specifically enriched in central nervous system neurons, its nuclear envelope localization indicates that D-aminoacyl-tRNA editing may be vital for the survival of neurons under high concentration of D-amino acids.     

Occurrence of free D-amino acids and aspartate racemases in hyperthermophilic archaea.

The occurrence of free D-amino acids and aspartate racemases in several hyperthermophilic archaea was investigated. Aspartic acid in all the hyperthermophilic archaea was highly racemized. The ratio of D-aspartic acid to total aspartic acid was in the range of 43.0 to 49.1%. The crude extracts of the hyperthermophiles exhibited aspartate racemase activity at 70 degrees C, and aspartate racemase homologous genes in them were identified by PCR. D-Enantiomers of other amino acids (alanine, leucine, phenylalanine, and lysine) in Thermococcus strains were also detected. Some of them might be by-products of aspartate racemase. It is proven that D-amino acids are produced in some hyperthermophilic archaea, although their function is unknown.