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.     

UDP-N-acetylmuramylpentapeptide as acceptor in murein biosynthesis in Escherichia coli membranes and ether-permeabilized cells

Two widely used in vitro systems of Escherichia coli capable of synthesizing murein were evaluated by using high-pressure liquid chromatography for murein analysis. Comparison of the composition of murein synthesized by either a membrane preparation or ether-treated cells with native murein revealed that both in vitro systems failed to synthesize murein that was identical to murein formed in vivo. Furthermore, neither system attached the lipoprotein to the murein. Ether-treated cells, however, were superior to the membrane preparation in catalyzing the formation of the remarkable A2pm-A2pm cross-linkage. In both systems an atypical transpeptidation reaction was found to take place in which exogenously supplied UDP-N-acetylmuramylpentapeptide was directly linked to the murein without participation of the bactoprenol lipid carrier. The direct transpeptidation yields preferentially trimeric peptide bridges with the UDP-linked muramylpentapeptide serving as the acceptor.     

Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli.

Certain D-amino acids, such as D-methionine and D-cystine, were incorporated into cells of Escherichia coli under conditions inhibiting protein and cell wall synthesis. Part of the radioactivity of D-14C-amino acids incorporated into the cells was found in the isolated cell wall peptidoglycan. A covalent linkage between the amino group of the D-amino acids and the peptidoglycan was presumed to be the main cause of the binding of the D-amino acids to peptidoglycan, because the amino group of the D-amino acids in the incorporation product was substituted. Whether the carboxyl terminus was substituted was unknown. The formation of the D-amino acid-peptidoglycan linkage was insensitive to beta-lactam antibiotics such as benzylpenicillin and ampicillin (500 micrograms/ml) and therefore was not due to the reaction of DD-transpeptidation which is involved in the biosynthesis of peptidoglycan. The D-amino acids also strongly inhibited the formation of peptidoglycan-bound lipoprotein in the E. coli cells. The results may suggest the correlation between binding of D-amino acid to peptidoglycan and inhibition of formation of the bound form of lipoprotein.     

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.     

Effect of cell membrane of Agrobacterium radiobacter on enhancing D-amino acids production from racemic hydantoins

An interesting phenomenon was observed that the existence of the intact cell membrane can enhance the D-amino acids production from D,L-5-substituted hydantoins by reacting with the whole cells of Agrobacterium radiobacter. Two intracellular enzymes were involved in the reaction process. The first enzyme D-hydantoinase converted hydantoins to carbamoyl derivatives which were further converted to D-amino acids by D-amidohydrolase. The amount of D-amino acids produced from hydantoins by the intact cells were 1.8–2.4 fold higher than the toluene treated cells. In addition, by using the intact cells the amount of D-amino acids produced from hydantoins was about 10 fold higher than that produced directly from carbamoyl derivatives. The relatively lower permeability of cell membrane to the reaction intermediate carbamoyl derivatives was confirmed by a simple mathematical model to be the main factor for the better performance of the intact cells for D-amino acid production. Besides, the low intracellular enzymes activities also contributed to the effect of intact cell membrane on enhancing the D-amino acid production.     

Incorporation of acyl moieties of phospholipids into murein lipoprotein in intact cells of Escherichia coli by phospholipid vesicle fusion.

The biosynthesis of the acyl moieties in murein lipoprotein was studied by fusion of [3H]palmitate-labeled phospholipid vesicles with intact cells of an fadD mutant of Escherichia coli. A linear increase in the incorporation of [3H]palmitate radioactivity into both the ester- and amide-linked fatty acids in lipoprotein was observed during a 3-h chase after the fusion. Addition of chloramphenicol completely prevented the incorporation of [3H]palmitate from phospholipids to lipoprotein. These results strongly support our hypothesis that the acyl moieties in phospholipids are the precursors for the fatty acids in murein lipoprotein of E. coli. Among the major glycerophosphatides in E. coli, no specificity was observed regarding the efficacy of the donor.     

ld-Carboxypeptidase activity in Escherichia coli

The activities of the LD-carboxypeptidases of Escherichia coli K 12 and of a mutant strain 155 with reduced activities were studied with the aid of ether treated cells. Evidence was obtained that was consistent with the suggestion that in both strains two LD-carboxypeptidase activities are present. Activity I degrades the nucleotide activated precursor UDP-MurNAc-tetrapeptide and activity II splits off D-alanine residues from position 4 of the peptide subunits in the nascent murein. In the mutant strain activity I is reduced 10fold compared with strain K 12, whereas activity II is not affected. The two activities could be distinguished with regard to their sensitivity to D-amino acids and the beta-lactam antibiotic thienamycin.     

氨基酸的手性与蛋白质生物合成

生命体中行使生物学功能的重要大分子蛋白质,由其基本单位氨基酸组成. 除甘氨酸外,其余19种常见氨基酸均具有手性,且均为L-构型,称为氨基酸的纯手性（homochirality,或称同手性）.这个现象长久以来困扰着科学家们. 本文简要综述了目前对纯手性起源的一些假说,D-氨基酸在生命体中的存在和可能的作用,以及D-氨基酸在蛋白质合成这个重要过程中的特性,包括D-氨基酸的氨酰化和在新生肽链的掺入. D-氨基酸的研究,让人们对生命有了更深入的认识,为疾病、制药等领域提供了新的思路,也为生命科学的基础研究提供了新的理论支撑和研究方向.     

Colicin M is an inhibitor of murein biosynthesis.

Colicin M inhibited the incorporation of DL + meso-2,6-diamino[3,4,5-3H]pimelic acid into the murein (peptidoglycan) of growing cells of Escherichia coli W7 dap lys. The inhibition of the UDP-N-acetylmuramyl pentapeptide-dependent incorporation of UDP-N-acetyl-D-[U-14C]glucosamine into isolated cell envelopes indicated interference with a late step of murein biosynthesis. After the inhibition of murein biosynthesis, cells lysed, and they released lysis products of murein. In vitro, the murein biosynthesis of colicin M-tolerant mutants (tolM) was inhibited by colicin M. Therefore, tolerance is probably conferred by an impaired uptake of an altered fixation close to the target site and not by a mutation of the target itself. Preliminary studies with beta-lactam antibiotics and with mutants in penicillin-binding proteins did not reveal a specific enzymatic step inhibited by colicin M. The unique action among the colicins renders colicin M a potentially useful tool for studying murein biosynthesis.     

Penicillin-binding proteins 4 and 5 of Haemophilus influenzae are involved in cell wall incorporation

Abstract Permeabilized cells of Haemophilus influenzae incorporate wall precursors into murein material in an ampicillin-sensitive reaction. In resistant transformants that contain the low antibiotic affinity penicillin-binding proteins (PBPs) 4 and 5, the sensitivity of this incorporation reaction to ampicillin is proportionally lower, suggesting a catalytic role for these proteins in wall synthesis. We conclude that, analogous to the reaction in Escherichia coli , PBPs 4 and 5 of H. influenzae have transpeptidase activity.     

Murein segregation in Escherichia coli.

Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.     

Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes

The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.     

A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli.

The first gene of a family of prokaryotic proteases with a specificity for L,D-configured peptide bonds has been identified in Escherichia coli. The gene named ldcA encodes a cytoplasmic L, D-carboxypeptidase, which releases the terminal D-alanine from L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine containing turnover products of the cell wall polymer murein. This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary growth phase. Owing to a defect in muropeptide recycling the unusual murein precursor uridine 5'-pyrophosphoryl N-acetylmuramyl-tetrapeptide accumulates in the mutant. The dramatic decrease observed in overall cross-linkage of the murein is explained by the increased incorporation of tetrapeptide precursors. They can only function as acceptors and not as donors in the crucial cross-linking reaction. It is concluded that murein recycling is a promising target for novel antibacterial agents.     

Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain D-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. D-amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how D-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. D-amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that D-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that D-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of D-amino acids in preventing device-related infections, we have shown that surfaces impregnated with D-amino acids were effective in preventing biofilm growth.     

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.     

Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli.

The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process.     

Novel jadomycins: incorporation of non-natural and natural amino acids

Electrospray ionization mass spectrometry of extracts from Streptomyces venezuelae ISP5230 cultures grown on chemically synthesized non-natural L-amino acids, D-amino acids or any of the 20 natural amino acids demonstrated incorporation of the amino acid into a jadomycin B analogue.     

The design of potent and selective inhibitors of thrombin utilizing a piperazinedione template: part 2.

Potent and selective thrombin inhibitors have been prepared with a piperazinedione template and L-amino acids. Likewise, incorporation of D-amino acids led to potent inhibitors with a novel mode of binding. Herein, the structure activity relationships and structural aspects of these compounds will be described.     

Theoretical approaches to D-amino acid oxidase

Several substrates and roles have been proposed for D-amino acid oxidase (E.C. 1.4.3.3.); however, there is no proof that they possess the required characteristics to account for the ubiquity, large amounts and great activity of the enzyme as found in diverse cells and tissues. Based on the similar stereoposition of identically charged atoms and lateral side chain (R) with respect to the alpha-hydrogen atoms in beta-sheet conformation and in D-amino acids, it is proposed that its substrates may include several membrane-related proteins, partially in beta-sheet conformation, whose alpha-hydrogen atoms would be the real object of D-amino acid oxidase catalysis. A monooxygenase-like enzymatic activity of D-amino acid oxidase with these novel substrates is considered, for which the final products are hypothesized to be protein alpha-carbon hydroxyls resulting from the incorporation of one atom of oxygen into the substrate, the other being reduced to water. Alternatively, it is also proposed that D-amino acid oxidase (and possibly other monooxygenase enzymes) would have a hydroperoxide-synthetase activity. In this case, protein alpha-carbon hydroperoxide and not water, but another reduced molecule, would be the final products. The new enzymatic performances of D-amino acid oxidase and the possible role of its potential final products in redox and other biochemical processes are discussed.     

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.