P-hydroxybenzoate Hydroxylase Research Articles

A New Enzyme Species of p-Hydroxybenzoate Hydroxylase during Oxygenating Process Observed by the Stopped-flow Method

Journal Article A New Enzyme Species of p-Hydroxybenzoate Hydroxylase during Oxygenating Process Observed by the Stopped-flow Method Get access Naoki Higashi, Naoki Higashi Department of Agricultural Chemistry, The University of Tokyo, Tokyo, 113, Japan Search for other works by this author on: Oxford Academic Google Scholar Hirofumi Shoun, Hirofumi Shoun Department of Agricultural Chemistry, The University of Tokyo, Tokyo, 113, Japan Search for other works by this author on: Oxford Academic Google Scholar Keiji Yano, Keiji Yano Department of Agricultural Chemistry, The University of Tokyo, Tokyo, 113, Japan Search for other works by this author on: Oxford Academic Google Scholar Kei Arima Kei Arima Department of Agricultural Chemistry, The University of Tokyo, Tokyo, 113, Japan Search for other works by this author on: Oxford Academic Google Scholar Agricultural and Biological Chemistry, Volume 36, Issue 6, 1 June 1972, Pages 1081–1084, https://doi.org/10.1080/00021369.1972.10860371 Published: 01 June 1972 Article history Received: 26 February 1972 Published: 01 June 1972

Interactions of substrate and non-substrate effectors with p-hydroxybenzoate hydroxylase from [formula omitted

3,4-Dihydroxybenzoate (3,4-DOHB), 2,4-dihydroxybenzoate (2,4-DOHB), and benzoate facilitate the interaction of p-hydroxybenzoate hydroxylase with TPNH. The two dihydroxybenzoate effectors form 1:1 complexes with the enzyme, inducing large spectral perturbations and fluorescence quenching. The dissociation constants for 2,4-DOHB and 3,4-DOHB are 0.15 and 0.50 mM respectively. During the reaction of enzyme with TPNH and oxygen, all the 2,4-DOHB, <5% of the benzoate, and none of the 3,4-DOHB is hydroxylated.

Mechanism of Action of p-Hydroxybenzoate Hydroxylase from Pseudomonas putida: III. THE ENZYME-SUBSTRATE COMPLEX

Abstract The mechanism of action of p-hydroxybenzoate hydroxylase from Pseudomonas putida, strain M-6, has been investigated. The aromatic substrate analogues, benzoate, p-fluorobenzoate, p-chlorobenzoate, p-nitrobenzoate, p-aminobenzoate, and 6-hydroxynicotinate, are found to be competitive inhibitors. This finding differs from the previously reported noncompetitive behavior in a different buffer system. The optical activity of the enzyme-inhibitor complex has been studied. From the kinetic and circular dichroism (CD) measurements, we have found that the carboxyl moiety is necessary and sufficient for the enzyme-substrate binding, whereas the hydroxyl group alone will not lead to binding. There are two classes of inhibitory analogues: one causes changes in CD spectra of the enzyme similar to those evoked by the substrate, and the other does not cause significant changes. The results indicate that more than one mode of enzyme-inhibitor interaction is involved. The CD of the enzyme-NADPH complex under anaerobic conditions suggests that the oxidized enzyme and reduced pyridine nucleotide form a complex, both in the absence and presence of the substrate, p-hydroxybenzoate. Furthermore, evidence for a ternary complex is given.

Catabolism of Aromatic Compounds by Micro-Organisms

This chapter discusses the catabolism of aromatic compounds by micro-organisms, and reviews (1) enzymic degradations of di- and trihydroxy phenols, (2) reactions converting aromatic compounds into ring-fission substrates, and (3) regulation of catabolic reaction sequences. Ortho-Fission pathways of catechol and protocatechuate as well as meta-fission pathways of catechols are discussed. Important information relating to meta fission has emerged from studies of the microbial degradation of steroids, and an outline of some of these reactions is also given. The enzymic hydroxylation of phenylalanine has been extensively investigated. The system resembles p-hydroxybenzoate hydroxylase except that the natural cofactor is dihydrobiopterin and not FAD. Pteridines resemble flavins in chemical structure and in their enzymic reactions, but they serve only as electron carriers for hydroxylations and not in the normal electron-transport systems. In both the mammalian and pseudomonad systems, hydroxylation of phenylalanine proceeds by a mechanism, which has been termed the “NIH shift”. The chapter also discusses the bacterial metabolism of gentisates, metabolism of thymol, the oxidation of phenylpropanoid structures arising from Lignins, degradation of α-conidendrin, and regulation of ortho-fission pathways.

Regulation of Synthesis of Early Enzymes of p-Hydroxybenzoate Pathway in Pseudomonas putida

Abstract The first two steps in the dissimilation of p-hydroxybenzoate through the β-ketoadipate pathway are (a) conversion of the primary substrate to protocatechuate by a monooxygenase, p-hydroxybenzoate hydroxylase, and (b) conversion of protocatechuate to β-carboxy-cis,cis-muconate by a dioxygenase, protocatechuate oxygenase. p-Hydroxybenzoate hydroxylase is specifically induced by its substrate. Protocatechuate oxygenase can be induced either by its substrate or by p-hydroxybenzoate. Exposure of cells to p-hydroxybenzoate therefore elicits simultaneous but independent inductions of both enzymes. Tests with a wide range of other aromatic acids showed that the specificity of induction of both enzymes is extremely high. No other effective inducers of protocatechuate oxygenase were discovered. A few of the compounds tested could induce slight activity of p-hydroxybenzoate hydroxylase, but the levels in all cases were less than 7% of that induced by p-hydroxybenzoate. The kinetic data of induction by p-hydroxybenzoate and by protocatechuate differ. Protocatechuate elicits immediate enzyme synthesis, whereas there is a lag of approximately 20 min before enzyme synthesis in response to p-hydroxybenzoate begins.

Kinetic studies on the reaction mechanism of p-hydroxybenzoate hydroxylase

Kinetic studies on the reaction mechanism of p-hydroxybenzoate hydroxylase

A non-substrate effector of p-hydroxybenzoate hydroxylase

A substrate analogue, 6-hydroxynicotinate (6-OHNA), facilitates reduction of the enzyme bound flavin by TPNH but is not hydroxylated in the reoxidation of the enzyme by oxygen. Binding of 6-OHNA to oxidized p-hydroxybenzoate hydroxylase is accompanied by a perturbation of the visible spectrum and enhancement of the flavin fluorescence. Titrations using these properties indicate the formation of a 1:1 complex with a dissociation constant of 2.5 × 10 −4 M. At high concentrations of 6-OHNA, binding of 6-OHNA to additional site(s) has been observed. The 6-OHNA binding is competitive with p-hydroxybenzoate binding as indicated by the reversal of p-hydroxybenzoate quenching of flavin fluorescence on addition of 6-OHNA. The presence of the effector, 6-OHNA, is necessary for the catalytic oxidation of TPNH.

Kinetic studies on the ES-complex formation of p-hydroxybenzoate hydroxylase using the stopped-flow method.

Journal Article Kinetic Studies on the ES-complex Formation of p-Hydroxybenzoate Hydroxylase Using the Stopped-flow Method Get access NAOKI HIGASHI, NAOKI HIGASHI Search for other works by this author on: Oxford Academic PubMed Google Scholar HIROHUMI SHOUN, HIROHUMI SHOUN Search for other works by this author on: Oxford Academic PubMed Google Scholar KEITARO HIROMI, KEITARO HIROMI *the Department of Chemistry, Faculty of Science, Kyoto UniversityKyoto Search for other works by this author on: Oxford Academic PubMed Google Scholar KEIJI YANO, KEIJI YANO Search for other works by this author on: Oxford Academic PubMed Google Scholar KEI ARIMA KEI ARIMA Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 67, Issue 5, May 1970, Pages 749–752, https://doi.org/10.1093/oxfordjournals.jbchem.a129305 Published: 01 May 1970 Article history Received: 24 December 1969 Published: 01 May 1970

Studies on the Interaction of p-Hydroxybenzoate Hydroxylase with the Substrate and Substrate Analogues

Studies on the Interaction of p-Hydroxybenzoate Hydroxylase with the Substrate and Substrate Analogues

Bacterial p-Hydroxybenzoate Hydroxylase

Screening for p-hydroxybenzoate-utilizing pseudomonads was carried out. Species belonging to Fluorescent group could grow at the expense of p-hydroxybenzoate as sole carbon source. Two species in Chromogenic group and three species in Achromogenic group could grow in the same manner. Specic and total activities of crude extracts differed from each other strain under cultural conditions employed. Ps. desmolytica IAM 1123 and Ps. desmolytica 4-B were selected as superior strains for the sources of p-hydroxybenzoate hydroxylase. The type and the activity of protocatechuate oxygenase were also investigated. Protocatechuate 3,4-oxygenase was found in the cells belonging to Fluorescent and Chromogenic groups whereas 4,5-oxygenase was found in Achromogenic group.

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

Crystallization and Properties of p-Hydroxybenzoate Hydroxylase from Pseudomonas putida

Abstract 1. The inducible p-hydroxybenzoate hydroxylase of Pseudomonas putida which catalyzes the hydroxylation of p-hydroxybenzoate to protocatechuate has been obtained in crystalline form as a protein homogeneous upon ultracentrifugation and electrophoresis. The molecular weight is estimated to be 83,600. 2. The enzyme contains approximately 1 mole of flavin adenine dinucleotide per mole of protein. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, serves as the hydrogen donor. 3. The enzyme is highly specific for p-hydroxybenzoate. Only four other aromatic compounds could be attacked at rates of less than 5% of the rate with p-hydroxybenzoate in all cases. 4. Under anaerobic conditions, the enzyme can catalyze the reduction of FAD by NADPH. This reaction requires the presence of the aromatic substrate to proceed at a significant rate. 5. The absorption spectrum of the enzyme in the visible region is slightly modified by the presence of the aromatic substrate.