Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid

Lignin and wheat bran represent renewable feedstocks for generation of useful and value-added compounds such as vanillin (a popular flavoring agent) and cis,cis-muconic acid (ccMA, a building block for the synthesis of plastic materials). In the present work, we report on the setup of an efficient and green process for producing such valuable compounds based on (a) the optimization of the extraction procedures for vanillin from lignin and ferulic acid from wheat bran and (b) the genetic engineering of an Escherichia coli strain with up to three plasmids differing in copy numbers to modulate the expression of up to seven recombinant enzymes. In detail, we used two sequential reactions catalyzed by the decarboxylase Fdc and the dioxygenase Ado to convert wheat bran-derived ferulic acid into vanillin: nature-identical vanillin was produced in one pot with a >85% yield in 20 h. Next, the dehydrogenase LigV, the demethylase VanAB, the decarboxylase AroY, and the dioxygenase C12O converted lignin-derived vanillin into ccMA with a >95% conversion yield and a productivity of 4.2 mg of ccMA/g of Kraft lignin in 30 min. Finally, when the optimized E. coli strain expressing all the abovementioned enzymes was used, ccMA was produced with a >95% conversion yield starting from ferulic acid in 10 h following product isolation, corresponding to 0.73 g of ccMA/g of ferulic acid, 1.4 g of ccMA/L, and 2.2 g of ccMA/g of wheat bran biomass. The optimized whole-cell system represents a sustainable and cost-competitive process for producing high value-added products from renewable resources

Evidence for the interaction of d-amino acid oxidase with pLG72 in a glial cell line.

Accumulating genetic evidence indicates that the primate-specific gene locus G72/G30 is related to schizophrenia: it encodes for the protein pLG72, whose function is still the subject of controversy. We recently demonstrated that pLG72 negatively affects the activity of human d-amino acid oxidase (hDAAO, also related to schizophrenia susceptibility), which in neurons and (predominantly) in glia is expected to catabolize the neuromodulator d-serine. The d-serine regulation mechanism relying on hDAAO-pLG72 interaction does not match with the subcellular localizations proposed for hDAAO (peroxisomes) and pLG72 (mitochondria). By using glioblastoma U87 cells transfected with plasmids encoding for hDAAO and/or pLG72 we provide convergent lines of evidence that newly synthesized hDAAO, transitorily present in cytosol before being delivered to the peroxisomes, colocalizes and interacts with pLG72 which we propose to be exposed on the external membrane of mitochondria. We also report that newly synthesized cytosolic hDAAO is catalytically active, and therefore pLG72 binding-and ensuing hDAAO inactivation-plays a protective role against d-serine depletion

Characterization of the human D-amino acid oxidase (hDAAO) - pLG72 complex involved in the onset of schizophrenia.

Schizophrenia is a chronic and severely debilitating psychiatric disorder affecting nearly 1% of the world’s population. In 2002, the new human gene G72, encoding for the pLG72 protein, and the gene encoding for DAAO have been genetically linked to the susceptibility to schizophrenia. A yeast two-hybrid screening experiment identified D-amino acid oxidase (DAAO) as a putative interacting partner of pLG72. DAAO is a FAD-containing flavooxidase that in brain is responsible for the elimination of D-serine, a co-agonist that binds to the glycine-site of the NMDA receptor. We recently demonstrated that pLG72 acts as “inactivator” of human DAAO and that the cellular concentration of D-serine depends on the expression of the active form of this flavooxidase. Based on these results, a molecular model for the onset of schizophrenia has been proposed: a decrease in pLG72 expression might yield an anomalous high level of hDAAO activity and therefore a decrease in the local concentration of D-serine, affecting glutamatergic neurotransmission mediated by NMDA receptor. The characterization of the complex is a challenging task, hardly feasible by high resolution techniques, since: 1) structural informations on pLG72 are lacking; 2) pLG72 is soluble only in the presence of mild denaturant; 3) no homologous protein has been structurally characterized so far. In this perspective, we have used low resolution strategies based on the coupling of classical biochemistry approaches (complementary proteolysis, cross-link) with mass spectrometric techniques, to characterize the pLG72-hDAAO complex. Results indicated that hDAAO exhibits different proteolysis profiles when isolated or in complex with pLG72, thus suggesting a conformational change upon binding the effector protein. Chemical cross-linking experiments will complement the proteolysis experiments providing with details the contact regions between hDAAO e pLG72

Role of tyrosine 238 in the active site of Rhodotorula gracilis D-amino acid oxidase - A site-directed mutagenesis study

Y238, one of the very few conserved residues in the active site of d-amino acid oxidases (DAAO), was mutated to phenylalanine and serine in the enzyme from the yeast Rhodotorula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active-site acid/base catalyst. Y238F and Y238S mutants exhibit a threefold slower turnover on d-alanine as substrate, which can be attributed to a slower rate of product release relative to the wild-type enzyme (a change of the rate constants for substrate binding was also evident). The Y238 DAAO mutants have spectral properties similar to those of the wild-type enzyme but the degree of stabilization of the flavin semiquinone and the redox properties in the free form of Y238S are different. The binding of the carboxylic acid competitive inhibitors and the substrate d-alanine are changed only slightly, suggesting that the overall substrate binding pocket remains intact. In agreement with data from the pH dependence of ligand binding and with the protein crystal structure, site-directed mutagenesis results emphasize the importance of residue Y238 in controlling access to the active site instead of a role in the substrate/ligand interaction

Characterization of the Covalently Bound Anionic Flavin Radical in Monoamine Oxidase A by Electron Paramagnetic Resonance

It was recently suggested that partially reduced monoamine oxidase (MAO) A contains an equilibrium mixture of an anionic flavin radical and a tyrosyl radical (Rigby, S. E.; et al. J. Biol. Chem. 2005, 280, 4627-4632). These observations formed the basis for a revised radical mechanism for MAO. In contrast, an earlier study of MAO B only found evidence for an anionic flavin radical (DeRose, V. J.; et al. Biochemistry 1996, 35, 11085-11091). To resolve the discrepancy, we have performed continuous-wave electron paramagnetic resonance at 94 GHz (W-band) on the radical form of MAO A. A comparison with D-amino acid oxidase (DAAO) demonstrates that both enzymes only contain anionic flavin radicals. Pulsed electron-nuclear double resonance spectra of the two enzymes recorded at 9 GHz (X-band) reveal distinct hyperfine coupling patterns for the two flavins. Density functional theory calculations show that these differences can be understood in terms of the difference at C8 of the isoalloxazine ring. DAAO contains a noncovalently bound flavin whereas MAO A contains a flavin covalently bound to a cysteinyl residue at C8. The similar electronic structures and hydrophobic environments of MAO and DAAO, and the similar structural motifs of their substrates suggest that a direct hydride transfer catalytic mechanism established for DAAO (Umhau, S.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12463-12468) should be considered for MAO

Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry

Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O-2-dependent oxidative deamination of D-enantiomer of amino acids generating alpha -keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37 degrees C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O-2-scavenger thus further hampering food deterioration

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution

Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover

Engineering the substrate specificity of D-amino-acid oxidase

The high resolution crystal structure of D-amino-acid oxidase (DAAO) from the yeast Rhodotorula gracilis provided us with the tool to engineer the substrate specificity of this flavo-oxidase. DAAO catalyzes the oxidative deamination of D-amino acids, with the exception of D-aspartate and D-glutamate (which are oxidized by D-aspartate oxidase, DASPO). Following sequence homology, molecular modeling, and simulated annealing docking analyses, the active site residue Met-213 was mutated to arginine. The mutant enzyme showed properties close to those of DASPO (e.g. the oxidation of D-aspartate and the binding of l-tartrate), and it was still active on D-alanine. The presence of an additional guanidinium group in the active site of the DAAO mutant allowed the binding (and thus the oxidation) of D-aspartate, but it was also responsible for a lower catalytic activity on D-alanine. Similar results were also obtained when two additional arginines were simultaneously introduced in the active site of DAAO (M213R/Y238R mutant, yielding an architecture of the active site more similar to that obtained for the DASPO model), but the double mutant showed very low stability in solution. The decrease in maximal activity observed with these DAAO mutants could be due to alterations in the precise orbital alignment required for efficient catalysis, although even the change in the redox properties (more evident in the DAAO-benzoate complex) could play a role. The rational design approach was successful in producing an enzymatic activity with a new, broader substrate specificity, and this approach could also be used to develop DAAO variants suitable for use in biotechnological applications

pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase - Effect on schizophrenia susceptibility

Human genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility