title: Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid-6-hydroxylase from Micrococcus.
abstract: 3-Hydroxybenzoic acid-6-hydroxylase from Micrococcus sp. was purified to homogeneity in a single step using the substrate-mediated interaction of the enzyme with blue-Sepharose. The enzyme was bound to the affinity matrix in the presence of 3-hydroxybenzoic acid and was eluted in its absence. The molecular weight of the purified enzyme is 70,000 with no subunit structure. The flavoenzyme required the exogenous addition of FAD for its complete activity and had a strict preference for NADH over NADPH. The activity of the enzyme was drastically inhibited by Cu2+ and Hg2+ and the inhibition was reversed by thiol reagents.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore.
authors.forename: S
authors.initials: S
authors.lastname: Rajasekharan
authors.forename: R
authors.initials: R
authors.lastname: Rajasekharan
authors.forename: C S
authors.initials: CS
authors.lastname: Vaidyanathan
mesh_terms: D002852:Chromatography, Ion Exchange; D062385:Hydroxybenzoates / Q000378:metabolism*; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology* / Q000254:growth & development; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D062385:Hydroxybenzoates; C032948:3-hydroxybenzoic acid; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase