The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.Work on the biosynthesis of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) in the archaea has revealed a number of surprises in terms of both the genes encoding the pathway enzymes and the pathway itself. Analysis of archaeal genomes has generally shown the absence of ribA, the gene encoding GTP cyclohydrolase II, the first enzyme in the presently established pathways to riboflavin in bacteria. In the archaea, this reaction may be catalyzed by at least two separate enzymes. The first enzyme, GTP cyclohydrolase III, produces 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5Ј-phosphate (compound 2 in Fig. 1) and inorganic pyrophosphate by hydrolysis of GTP (11). This intermediate is subsequently hydrolyzed to 2,5-diamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5Ј-phosphate (compound 3) by a currently unknown enzyme. Compound 3 is then converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (ARP, compound 6 in Fig. 1) by a dehydrogenase (MJ0671) (5), a deaminase, and a phosphatase following the eukaryotic pathway (13). The involvement of compound 2 does not occur in either the bacterial or the eukaryotic pathway, and the dehydrogenase and deaminase steps are in the same order as in the eukaryotic pathway, which is the reverse of that found in the bacterial pathway. The product of this series of reactions, ARP, is the precursor of F 420 and riboflavin (10). In the conversion of ARP to riboflavin in the archaea, there is a fundamental difference in the stereochemistry of the pentacyclic intermediate involved in the dismutation of 6,7-dimethyl-8-ribityllumazine into riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione in the last step of riboflavin biosynthesis (17).Two different groups of enzymes are known to be involved in the conversion of riboflavin first to FMN and then to FAD. In the first group, the reaction is catalyzed by FAD synthetase, a bifunctional enzyme (RibF or RibC) that first acts as a kinase converting riboflavin to FMN in the presence of ATP and then acts as a nucleotidyltransferase by using a second ATP to convert the FMN to FAD and PP i (8,21). In the other group, these reactions are catalyzed by two separate enzymes, riboflavin kinase (RibR, flavokinase, or FMN1) and FAD synthetase (FAD1 in yeast) (32). Enzymes homologous to the yeast flavokin...
N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 μm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 μm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 μm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 μm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure–activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.
Background: Fatty acid heme dioxygenases occur in eukaryotes, often associated with a cytochrome P450 that transforms the peroxide product. Results: Neighboring cyanobacterial genes, dioxygenase and catalase, are identified as linoleate 10S-dioxygenase and 10S-hydroperoxide lyase, respectively. Conclusion: These Nostoc hemoproteins show novel activities. Significance: Our results identify a heme dioxygenase ancestor and a catalase that substitutes in function for a cytochrome P450.
FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.
This review focuses on a group of heme peroxidases that retain the catalase fold in structure, yet show little or no reaction with hydrogen peroxide. Instead of a role in oxidative defense, generally these enzymes are involved in secondary metabolite biosynthesis. The prototypical enzyme is the catalase-related allene oxide synthase (cAOS), an enzyme that converts a specific fatty acid hydroperoxide to the corresponding allene oxide (epoxide). Other catalase-related enzymes form allylic epoxides, aldehydes or a bicyclobutane fatty acid. In all catalases, including these catalase relatives, a His residue on the distal face of the heme is absolutely required for activity. Its immediate neighbor in sequence as well as in three-dimensional space is conserved as Val in true catalases and changed to Thr in the fatty acid hydroperoxide-metabolizing enzymes. As explained herein, the Thr-His connection on the distal face of the heme is critical in switching the substrate specificity from H2O2 to the transformation of fatty acid hydroperoxide.
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