Performance evaluation on a wide set of matrix-assisted laser desorption ionization matrices for the detection of oligosaccharides in a high-throughput mass spectrometric screening of carbohydrate depolymerizing enzymes.

Compared to other analytical methods, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) presents several unique advantages for the structural characterization of degradation products of carbohydrates. Our final goal is to implement this technique as a high-throughput platform, with the aim of exploring natural bio-diversity to discover new carbohydrate depolymerizing enzymes. In this approach, a variety of carbohydrates will be used as enzymes substrates and MALDI-MS will be employed to monitor the oligosaccharides produced. One drawback of MALDI, however, is that the choice of the matrix is largely dependent on the chemical properties of the analyte. In this context, our objective in the present work was to find the smallest set of MALDI matrices able to detect chemically heterogeneous oligosaccharides. This was done through the performance evaluation of more than 40 MALDI matrices preparations. Homogeneity of analyte-matrix deposits was considered as a critical feature, especially since the final objective is to fully automate the analyses. Evaluation of the matrices was done by means of a rigorous statistical approach. Amongst all tested compounds, our work proposes the use of the DHB/DMA ionic matrix as the most generic matrix, for rapid detection of a variety of polysaccharides including neutral, anionic, methylated, sulfated, and acetylated compounds. The selected matrices were then used to screen crude bacterial incubation media for the detection of enzymatic degradation products.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450.

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450.

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the omega3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10muM of these two omega3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of omega3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.

Determination of polyunsaturated fatty acid monoepoxides by high performance liquid chromatography-mass spectrometry.

Despite the implication of polyunsaturated fatty acid monoepoxides in a large panel of biological effects, few methods allowing their separation in a single run are available. We describe here a simple method based on reversed-phase ion-pair high-performance liquid chromatography (RP-HPLC) and developed to successfully separate the various monoepoxides of eicosatrienoic, arachidonic, eicosapentaenoic and docosahexaenoic acids. These compounds were easily identified by liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure chemical ionisation owing to the volatility of counter-ion species. Compared to established methods, this new protocol proved its ability to totally resolve, in a single run, all of the different regioisomeric epoxides. In the long run, this method will demonstrate its efficacy to give insights into the cytochrome P450-dependent metabolism of polyunsaturated fatty acids (PUFAs) and the generation of physiologically active epoxy-derivatives.

Involvement of cytochrome P450 1A2 in the biotransformation of trans-resveratrol in human liver microsomes.

This study was aimed at identifying the isoform(s) of human liver cytochrome P450 (CYP) involved in the hepatic biotransformation of trans-resveratrol (trans-3,5,4'-trihydroxystilbene). Trans-resveratrol metabolism was found to yield two major metabolites, piceatannol (3,5,3',4'-tetrahydroxystilbene) and another tetrahydroxystilbene named M1. Trans-resveratrol was hydroxylated to give piceatannol and M1 with apparent K(m) of 21 and 31 microM, respectively. Metabolic rates were in the range 14-101 pmol min(-1) mg(-1) protein for piceatannol and 29-161 pmol min(-1) mg(-1) protein for M1 in the 13 human liver microsomes tested. Using microsomal preparations from different human liver samples, piceatannol and M1 formation significantly correlated with ethoxy-resorufin-O-deethylation (r(2) = 0.84 and 0.88, respectively), phenacetin-O-deethylation (r(2) = 0.92 and 0.94) and immuno-quantified CYP1A2 (r(2) = 0.85 and 0.90). Formation of these metabolites was markedly inhibited by alpha-naphthoflavone and furafylline, two inhibitors of CYP1A2. Antibodies raised against CYP1A2 also inhibited the biotransformation of trans-resveratrol. In addition, the metabolism of trans-resveratrol into these two metabolites was catalyzed by recombinant human CYP1A1, CYP1A2 and CYP1B1. Our results provide evidence that in human liver, CYP1A2 plays a major role in the metabolism of trans-resveratrol into piceatannol and tetrahydroxystilbene M1.

Medium-throughput profiling method for screening polysaccharide-degrading enzymes in complex bacterial extracts.

Polysaccharides are the most abundant and the most diverse renewable materials found on earth. Due to the stereochemical variability of carbohydrates, polysaccharide-degrading enzymes - i.e. glycoside hydrolases and polysaccharide lyases - are essential tools for resolving the structure of these complex macromolecules. The exponential increase of genomic and metagenomic data contrasts sharply with the low number of proteins that have ascribed functions. To help fill this gap, we designed and implemented a medium-throughput profiling method to screen for polysaccharide-degrading enzymes in crude bacterial extracts. Our strategy was based on a series of filtrations, which are absolutely necessary to eliminate any reducing sugars not directly generated by enzyme degradation. In contrast with other protocols already available in the literature, our method can be applied to any panel of polysaccharides having known and unknown structures because no chemical modifications are required. We applied this approach to screen for enzymes that occur in Pseudoalteromonas carrageenovora grown in two culture conditions.

Cytochromes P450 from family 4 are the main omega hydroxylating enzymes in humans: CYP4F3B is the prominent player in PUFA metabolism.

Human CYP450 omega-hydroxylases of the CYP4 family are known to convert arachidonic acid (AA) to its metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). This study deals with hydroxylations of four PUFAs, eicosatrienoic acid (ETA), AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) by either human recombinant CYP4s enzymes or human liver microsomal preparations. CYP4F3A and CYP4F3B were the most efficient omega-hydroxylases of these PUFAs. Moreover, the differences in the number of unsaturations of ETA, AA, and EPA allowed us to demonstrate a rise in the metabolic rate of hydroxylation when the double bond in 14-15 or 17-18 was missing. With the CYP4F enzymes, the main pathway was always the omega-hydroxylation of PUFAs, whereas it was the (omega-1)-hydroxylation with CYP1A1, CYP2C19, and CYP2E1. Finally, we demonstrated that the omega9 and omega3 PUFAs (ETA, EPA, and DHA) could all be used as alternative substrates in AA metabolism by human CYP4F2 and -4F3B. Thus, they decreased the ability of these enzymes to convert AA to 20-HETE. However, although ETA was the most hydroxylated substrate, EPA and DHA were the most potent inhibitors of the conversion of AA to 20-HETE. These findings suggest that some physiological effects of omega3 FAs could partly result from a shift in the generation of active hydroxylated metabolites of AA through a CYP-mediated catalysis.