flavin-adenine-dinucleotide and pipecolic-acid

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines L-proline, L-pipecolic acid and sarcosine, with optimal catalytic activity towards L-proline. The holoenzyme contains one FAD, FMN and ATP per alphabeta complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with L-proline, for which a bell-shaped dependence (pK(a) values 7.0 +/- 0.2 and 7.6 +/- 0.2) for k(cat)/K(m) as a function of pH was observed. The pH dependence of k(cat) is sigmoidal, described by a single macroscopic pK(a) of 7.7 +/- 0.1, tentatively attributed to ionization of L-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint potential of enzyme-bound FAD has a linear temperature dependence (- 3.1 +/- 0.05 mV x C degrees (-1)), and extrapolation to physiologic growth temperature for P. furiosus (100 degrees C) yields a value of - 407 +/- 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic L-proline dehydrogenase. Implications for its mechanism of action are discussed.

Topics: Amino Acid Sequence; Binding Sites; Dimethylglycine Dehydrogenase; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases Acting on CH-NH Group Donors; Pipecolic Acids; Proline; Proline Oxidase; Protein Subunits; Pyrococcus furiosus; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature

Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.

Topics: Amino Acid Sequence; Animals; Caenorhabditis elegans; Cell Line; Cloning, Molecular; DNA, Complementary; Flavin-Adenine Dinucleotide; Flavoproteins; Fluorescent Antibody Technique, Indirect; Humans; Kidney; Kinetics; Microbodies; Molecular Sequence Data; Oxidoreductases, N-Demethylating; Pipecolic Acids; Proline; Rabbits; Sarcosine; Sarcosine Oxidase; Sequence Alignment; Sequence Homology, Amino Acid; Subcellular Fractions