flavin-adenine-dinucleotide and 2-5-dihydroxybenzoic-acid

Corynebacterium glutamicum ATCC 13032 metabolizes 3-hydroxybenzoate via gentisate. We have now characterized the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase involved in the initial step of 3-hydroxybenzoate catabolism by this strain, a first 3-hydroxybenzoate 6-hydroxylase molecularly and biochemically characterized from a Gram-positive strain. The ncg12923 gene from Corynebacterium glutamicum ATCC 13032 was shown to encode 3-hydroxybenzoate 6-hydroxylase, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Ncgl2923 was expressed with an N-terminal six-His tag and purified to apparent homogeneity by Ni²(+)-nitrilotriacetic acid affinity chromatography. The purified H₆-Ncgl2923 showed a single band at apparent molecular mass of 49 kDa on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be most likely a trimer as determined by gel filtration chromatography. It had a specific activity of 6.92 ± 0.39 U mg⁻¹ against 3-hydroxybenzoate and with a K(m) value of 53.4 ± 4.7 μM using NADH as a cofactor. The product formed from the 3-hydroxybenzoate hydroxylation catalyzed by H₆-Ncgl2923 was identified by high-performance liquid chromatography as gentisate, a ring-cleavage substrate in the microbial aromatic degradation. The enzyme exhibited a maximum activity at pH 7.5 in phosphate buffer, and adding flavin adenine dinucleotide to a final concentration of 15 μM would enhance the activity by three-fold. Although this enzyme shares no more than 33% identity with any of reported 3-hydroxybenzoate 6-hydroxylases from Gram-negative bacterial strains, there is little difference in subunit sizes and biochemical characteristics between them.

Topics: Amino Acid Sequence; Buffers; Chromatography, Affinity; Chromatography, Gel; Coenzymes; Corynebacterium glutamicum; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Gentisates; Hydrogen-Ion Concentration; Hydroxybenzoates; Kinetics; Mixed Function Oxygenases; Molecular Sequence Data; Molecular Weight; Protein Multimerization; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid

We isolated 3-hydroxybenzoate-6-hydroxylase (E.C.1.14.13.), an inducible enzyme that catalyzed the para-hydroxylation of 3-hydroxybenzoate (3-HBA) to 2,5-dihydroxybenzoate, from Klebsiella pneumoniae. Although the enzyme was found to be mainly induced by its substrate, a coordinated induction of 3-hydroxybenzoate hydroxylase and gentisate dioxygenase was also observed in the presence of the product of the reaction. The purified enzyme was a monomer with a molecular mass of 42,000. It contained FAD as a prosthetic group, utilized NADH or NADPH with similar efficiencies and its activity was inhibited by Cu2+, Fe2+ and Hg2+. Other properties, such as induction mechanism and kinetic parameters were also studied. Moreover, for the first time the amino acid composition of a 3-hydroxybenzoate-6-hydroxylase was determined.

Topics: Amino Acids; Bacterial Proteins; Cations, Divalent; Chromatography, Ion Exchange; Diethyl Pyrocarbonate; Dioxygenases; Enzyme Induction; Flavin-Adenine Dinucleotide; Gentisates; Hydroxybenzoates; Kinetics; Klebsiella pneumoniae; Mixed Function Oxygenases; Molecular Weight; NAD; NADP; Oxygenases

Topics: Biochemical Phenomena; Biochemistry; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Gentisates; Homogentisic Acid; Liver; Oxidoreductases; Rats; Research; Riboflavin Deficiency; Spectrum Analysis; Thiamine Deficiency; Ultracentrifugation