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.     

Post-transfer editing mechanism of a D-aminoacyl-tRNA deacylase-like domain in threonyl-tRNA synthetase from archaea

To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD). High-resolution crystal structures of Pab-NTD with pre- and post-transfer substrate analogs and with L-serine show mutually nonoverlapping binding sites for the seryl moiety. Although the pre-transfer editing is excluded, the analysis reveals the importance of main chain atoms in proper positioning of the post-transfer substrate for its hydrolysis. A single residue has been shown to play a pivotal role in the inversion of enantioselectivity both in Pab-NTD and DTD. The study identifies an enantioselectivity checkpoint that filters opposite chiral molecules and thus provides a fascinating example of how nature has subtly engineered this domain for the selection of chiral molecules during translation.     

A biosensor for all D-amino acids using evolved D-amino acid oxidase

Determination of the D-amino acid content in foods and in biological samples is a very important task. In order to achieve this goal we developed a biosensor employing the flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis. To produce a device in which the D-amino acid composition does not alter the results, both the wild-type and a number of mutants obtained by rational design and directed evolution approaches were used. An analysis of D-amino acid oxidase mutants activity on D-amino acid mixtures containing various ratios of neutral, acidic, and basic substrates identified the Amberzyme-immobilized T60A/Q144R/K152E and M213G mutants as the best choice: their response shows an only limited dependence on the solution composition when at least 20% of the D-amino acid is made up of D-alanine (standard error is approximately 5-9%). This is the first report, to our knowledge, demonstrating that the entire D-amino acid content can be determined by using a screen-printed electrode amperometric biosensor, with a detection limit of 0.25 mM and a mean response time of 10-15 min. The D-amino acid assay based on R. gracilis DAAO-biosensor is inexpensive, simple to perform, and rapid: the D-amino acid concentration of a variety of biological samples can be investigated using this assay.     

Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).     

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.     

Lifetime of the purple intermediate of D-amino acid oxidase under anaerobic conditions.

The lifetime of the purple intermediate formed from D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] and a neutral D-amino acid under anaerobic conditions was measured with a series of neutral D-amino acids. The lifetime increases with increase in the number of carbons in the side chain of the amino acid up to 4-5 atoms and then decreases with further increase in the number of carbons. This suggests that the hydrophobicity of the alkyl group of the neutral D-amino acid determines the lifetime of the purple intermediate unless a steric effect occurs.     

Detection and substrate selectivity of new microbial D-amino acid oxidases

In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R. gracilis) and D-proline (pig kidney). The D-amino oxidases from F. oxysporum and V. luteoalbum were able to react with the industrially important substrate cephalosporin C although the D-amino acid oxidase from T. variabilis was at least about 20-fold more active with this substrate.As the results of our studies, a reliable oxidase assay was developed, allowing high throughput screening in a microplate reader. Furthermore, three new microbial D-amino acid oxidase-producers with interesting broad substrate specificities were introduced in the field of biotechnology.     

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。     

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.     

Effect of D-amino acids on structure and synthesis of peptidoglycan in Escherichia coli.

Growth of Escherichia coli in the presence of certain D-amino acids, such as D-methionine, results in the incorporation of the D-amino acid into macromolecular peptidoglycan and can be lethal at high concentrations. Previous studies suggested that incorporation was independent of the normal biosynthetic pathway. An enzymatic reaction between the D-amino acid and macromolecular peptidoglycan was proposed as the mechanism of incorporation. The application of more advanced analytical techniques, notably high-pressure liquid chromatography, revealed that the presence of a D-amino acid susceptible to incorporation induced a multiplicity of alterations in peptidoglycan metabolism. Results derived basically from the study of samples treated with D-Met, D-Trp, and D-Phe indicated that the incorporation of a D-amino acid results in the accumulation of two major new muropeptides whose general structures most likely are GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa and GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-Ala-GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa, where D-aa represents a residue of the added D-amino acid. Resting cells are proficient in the incorporation of D-amino acids and can reach peptidoglycan modification levels comparable to those in growing cells. Under our conditions, D-amino acids had no apparent effect on growth or morphology but caused a severe inhibition of peptidoglycan synthesis and cross-linking, possibly leading to a reduction in the amount of peptidoglycan per cell. The properties of the reaction support the involvement of a penicillin-insensitive LD-transpeptidase enzyme in the synthesis of modified muropeptides and a possible inhibitory action of D-amino acids on high-molecular-weight penicillin-binding proteins.     

D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal,Ornithorhynchus anatinus,the Australian platypus

The C-type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D-amino acid at position 2 in the primary structure. Peptides containing a D-amino acid have been found in lower forms of organism, but this report is the first for a D-amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L-amino acid residue in the protein to the D-configuration.     

Stereoselectivity of 1-aminocyclopropanecarboxylate malonyltransferase toward stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid

A malonyltransferase isolated from mungbean (Vigna radiata L.) hypocotyls catalyzed the malonylation of both 1-aminocyclopropane-1-carboxylic acid (ACC) and D-amino acids. The possibility that ACC was recognized by the enzyme as a D-amino acid was investigated by examining the efficiencies of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) serving as substrates of malonyltransferase and as inhibitors of ACC malonyltransferase. Although all four isomers were malonylated by the enzyme and competitively inhibited the malonylation of ACC to N-malonyl-ACC, (1R,2S)-AEC and (1R,2R)-AEC, both of which have an R-configuration as a D-amino acid, had lower Km and Ki values (0.1 to 0.2 mM) than their enantiomers, (1S,2R)-AEC (Km and Ki values were about 1 mM) and (1S,2S)-AEC (Km and Ki values were higher than 10 mM), which have an S-configuration as an L-amino acid. Similarly, (R)-isovaline (2-amino-2-methylbutanoic acid), which has an R-configuration as a D-amino acid, inhibited more effectively the enzymatic conversion of ACC to malonyl-ACC than did (S)-isovaline, which has an S-configuration as an L-amino acid. In mungbean hypocotyls (1R,2S)-AEC and (1R,2R)-AEC were also more efficiently converted into malonyl conjugates and more efficiently inhibited the conversion of radioactive ACC into malonyl-ACC than their enantiomers, although the differences in efficiency among stereoisomers were smaller in hypocotyls than in enzymatic reactions. These results suggest that ACC is recognized by the enzyme as a D-amino acid.     

荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。     

Analyzing the D-amino acid content in biological samples by engineered enzymes

The aim of our present research is to produce mutant forms of D-amino acid oxidase from Rhodotorula gracilis in order to determine D-amino acid content in different biological samples. During the past few years, our group has produced yeast D-amino acid oxidase variants with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all D-amino acids) both by rational design and directed evolution methods. Now, the kinetic constants for a number of amino acids (even for unnatural ones) of the most relevant D-amino acid oxidase variants have been investigated. This information constitutes the basis for considering potential analytical applications in this important field.     

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.     

Factors controlling the level and determination of D-amino acids in the urine and plasma of laboratory rodents

Summary Unambiguous methodologies were developed for the accurate and reproducible determination of specific D-amino acids in the physiological fluids of common laboratory rodents. Depending on the strain of rodent and the type of amino acid examined, excreted D-amino acids ranged from the low percent levels to over 40 percent of the total specific amino acid level. Relative plasma levels tended to be considerably lower, typically an order of magnitude less. A number of factors were found to alter the relative amounts of excreted D-amino acids. This included: diet, age, pregnancy, advanced cancer, and antibiotics. The two factors that seemed to result in substantially lower levels of excreted D-amino acids were fasting and young age. Pregnancy was the only factor that consistently resulted in higher relative D-amino acid excretion. Much of the observed data are believed to be related to the efficiency with which the kidney reabsorbs L-amino acids. No claims are made as to the meaning and/or importance of free D-amino acids in regards to pathology, age, clinical usefulness and so forth. However, a knowledge of normal D-amino acid levels and dynamics is necessary before it is possible to identify perturbations caused by either natural or pathological conditions. The techniques are now available that should allow these topics to be addressed properly.On leave from Kyungpook National University in Korea.On leave from Institute of Physical Sciences, Polish Academy of Sciences.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

Second lytic target of beta-lactam compounds that have a terminal D-amino acid residue

A new biochemical mechanism of lysing bacterial cells by treatment with certain beta-lactam compounds that possess a terminal D-amino acid moiety in their side chain was demonstrated. The two functions of the molecule, the beta-lactam and terminal D-amino acid moiety, are both involved in the activity of lysing gram-negative bacteria, which is characterized by very rapid lysis of the cells in the first few hours after their contact with the compound. This mechanism was proved by studies on one such compound, named MT-141, which contains a terminal D-cysteine moiety with free amino and carboxyl groups in the 7 beta-side chain of the 7 alpha-methoxy-cephalosporin skeleton. This compound bound to the cell-wall peptidoglycan of Escherichia coli through the D-amino group of its terminal D-amino acid moiety and this seemed to cause rapid cell lysis. Both activities, of binding to peptidoglycan and of causing rapid cell lysis, were inhibited by certain D-amino acids, but not by L-amino acids. Mutants were isolated that had simultaneously gained decreased sensitivity to this kind of beta-lactam compound and supersensitivity to globomycin, an inhibitor of formation of lipoproteins which function in linking the peptidoglycan to the outer membrane. These results suggest that binding of the terminal D-amino acid moiety of the beta-lactam compound to peptidoglycan somehow influences formation of the linkage between the outer membrane and the peptidoglycan and consequently enhances the cell lytic activity of the beta-lactam portion of the molecule.     

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Background  

Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An^LC_L domain catalyzes a peptide bond between two L-amino acids, a^DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β -hydroxy-carboxylic acid (typically a β -hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.