flavin-adenine-dinucleotide and anthranilic-acid

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (

Topics: Apoenzymes; Bacterial Proteins; Benzoic Acid; Biocatalysis; Desulfovibrio vulgaris; Electron Transport; Enterobacter cloacae; Flavin-Adenine Dinucleotide; Flavodoxin; Holoenzymes; Models, Molecular; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Nitroreductases; ortho-Aminobenzoates; Oxidation-Reduction; Oxidoreductases; Pyrococcus furiosus; Recombinant Fusion Proteins; Recombinant Proteins; Silent Mutation; Thermus thermophilus

Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH(2)-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the anthranilate degradation gene cluster in Geobacillus thermodenitrificans NG80-2 were functionally characterized in vitro. GTNG_3159 produces FAD to be reduced by GTNG_3158 and the reduced FAD (FADH(2)) is utilized by GTNG_3160 to convert anthranilate to 3-hydroxyanthranilate (3-HAA), which is further degraded to acetyl-CoA through a meta-cleavage pathway also encoded in the gene cluster. Utilization of this pathway for the degradation of anthranilate and tryptophan by NG80-2 under physiological conditions was confirmed by real-time RT-PCR analysis of representative genes. This is believed to be the first time that the degradation pathway of anthranilate via 3-HAA has been characterized in a bacterium. This pathway is likely to play an important role in the survival of G. thermodenitrificans in the oil reservoir conditions from which strain NG80-2 was isolated.

Topics: Bacterial Proteins; Cloning, Molecular; Escherichia coli; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Genes, Bacterial; Geobacillus; Hydrolases; Metabolic Networks and Pathways; Multigene Family; Nucleotidyltransferases; ortho-Aminobenzoates; Oxidoreductases; Phosphotransferases (Alcohol Group Acceptor); Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Temperature

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.

Topics: Catalytic Domain; Crystallography, X-Ray; D-Amino-Acid Oxidase; Dimerization; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Models, Molecular; ortho-Aminobenzoates; Protein Conformation; Rhodotorula; Structural Homology, Protein; Yeasts