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.     

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.     

Structural adaptation of the glycophorin A transmembrane homodimer to D-amino acid modifications

Protein-protein recognition is an essential process in life. The chemistry of these kind of interactions is predominantly stereospecific (i.e. receptor-ligand, antibody-hapten binding). Here, we investigated whether the hydrophobic nature of the membrane affects this stereospecificity. To this end, we synthesized a diastereomer analogue (2D-GPA) of the glycophorin A transmembrane helix, with two l-valine residues replaced by their d-enantiomer. This ensures a disruption of the secondary structure. We investigated the ability of the diastereomer peptide to recognize the GPA chimera in the ToxR homodimer reporting system, in vivo. The peptide demonstrated a dose-dependent dominant negative effect on the GPA transmembrane in the bacterial ToxR system, suggesting a wild-type like interaction. This result was corroborated in vitro by fluorescence energy transfer between 2D-GPA and all-l GPA. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blot confirmed that ToxR GPA receptor levels are not affected by the presence of the exogenous peptide. In order to understand the structural basis for heterodimer formation, homodimer and heterodimer structures, based on the NMR 3D structure of GPA, were subjected to a molecular dynamics simulation. The resulting heterodimer structure maintained most of the original inter-helical interactions, and its structure is similar to that of the homodimer. We postulate that the need to satisfy all H-bonds can compensate for the structural strain induced by the presence of the d-amino acid residues.     

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.     

Evolution of translational initiation: new insights from the archaea

Recent in silico and experimental data have shed new light on the mechanism and components of translational initiation in archaea. The available data about the structure of archaeal mRNAs, mRNA/ribosome interaction and archaeal translation initiation factors are reviewed and analyzed in the conceptual framework of the evolution of translational initiation. A model of the initiation step of translation in the Last Universal Common Ancestor of extant cells is presented and discussed.     

Synthesis of the translational apparatus is regulated at the translational level.

The synthesis of many mammalian proteins associated with the translational apparatus is selectively regulated by mitogenic and nutritional stimuli, at the translational level. The apparent advantages of the regulation of gene expression at the translational level are the speed and the readily reversible nature of the response to altering physiological conditions. These two features enable cells to rapidly repress the biosynthesis of the translational machinery upon shortage of amino acids or growth arrest, thus rapidly blocking unnecessary energy wastage. Likewise, when amino acids are replenished or mitogenic stimulation is applied, then cells can rapidly respond in resuming the costly biosynthesis of the translational apparatus. A structural hallmark, common to mRNAs encoding many components of the translational machinery, is the presence of a 5' terminal oligopyrimidine tract (5'TOP), referred to as TOP mRNAs. This structural motif comprises the core of the translational cis-regulatory element of these mRNAs. The present review focuses on the mechanism underlying the translational control of TOP mRNAs upon growth and nutritional stimuli. A special emphasis is put on the pivotal role played by ribosomal protein S6 kinase (S6K) in this mode of regulation, and the upstream regulatory pathways, which might be engaged in transducing external signals into activation of S6K. Finally, the possible involvement of pyrimidine-binding proteins in the translational control of TOP mRNAs is discussed.     

Structural studies of the translational apparatus.

Significant progress is occurring at an accelerated rate in structural studies of ribosomes. A 3D cryoelectron microscopy map of the 70S ribosome from Escherichia coli is available at 15 A resolution and a combination of cryoelectron microscopy with X-ray crystallography has yielded a 9 A resolution map of the 50S subunit from Haloarcula marismortui, an archaebacterium. For eukaryotes, 3D cryomaps of the 80S ribosomes from yeast and from mammals have now been produced at resolutions in the range 20 to 30 A. The most ground-breaking results have been obtained from the 3D mapping of ligands in functional studies of prokaryotic ribosomes. These studies, which directly visualize the protein synthesis machine in action, have brought new excitement to a field that was relatively dormant during the past decade.     

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid‐cell zone for division. The universality of pattern‐forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time‐lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.     

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.     

D-amino acid oxidase in mouse liver--II

1. Activity of D-amino acid oxidase was detected in tissue extract of mouse liver by two sensitive spectrophotometric methods. 2. The activity was also detectable in extracts of the heart, but not of lung.     

First-principles molecular dynamics investigation of the D-amino acid oxidative half-reaction catalyzed by the flavoenzyme D-amino acid oxidase

Large-scale Car-Parrinello molecular dynamics simulations of D-alanine oxidation catalyzed by the flavoenzyme D-amino acid oxidase have been carried out. A model of the enzyme active site was built by starting from the enzyme X-ray structure, and by testing different subsystems comprising different sets of aminoacyl residues. In this process, the stability of the enzyme-substrate complex was taken as a measure of the accuracy of the model. The activated transfer of the amino acid alpha-hydrogen from the substrate to the flavin N5 position was then induced by constraining a suitable transfer reaction coordinate, and the free energy profile of the reaction was calculated. The evolution of electronic and structural properties of both enzyme-bound substrate and flavin cofactor along the reaction path is consistent with a hydride-transfer mechanism. The calculated free energy barrier for this process (13 kcal/mol) is in excellent agreement with the activation energy value derived from the experimentally determined rate constant for the corresponding enzyme-catalyzed reaction. The electronic distribution of the reduced flavin shows that the transferred electrons tend to be centered near the C4a position rather than delocalized over the flavin pyrimidine ring. This feature is mechanistically relevant in that such an electronic distribution may promote the subsequent enzyme-catalyzed reduction of molecular oxygen to yield hydrogen peroxide via a postulated flavin 4a-peroxide intermediate. These results also show that a first-principles molecular dynamics approach is suitable to study the mechanism of complex enzymatic processes, provided that a smaller, yet reliable, subsystem of the enzyme can be identified, and special computational techniques are employed to enhance the sampling of the reactive event.     

D-amino acid oxidase in mouse liver

1. An appreciable amount of D-amino acid oxidase was found in the extract of mouse liver by enzyme-linked immunosorbent assay (ELISA). 2. The content of the enzyme in the kidney and heart extracts was also measured by the assay.     

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.