Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens catalyzes in vivo the hydroxylation of p-hydroxybenzoate by molecular oxygen to form 3,4-dihydroxybenzoate. p-Mercaptobenzoate is also a substrate of the enzyme, but instead of being converted to the expected product, 3-hydroxy-4-mercaptobenzoate, the disulfide, 4,4'-dithiobisbenzoate, is formed. To find what mechanistic information this unusual reaction provided, steady state kinetic analyses, combined with rapid reaction studies of the changes in the enzyme-bound FAD, were carried out with the separate half-reactions involved in catalysis. Most of the kinetic measurements were made with a stopped-flow spectrophotometer designed for working anaerobically and connected on line to a minicomputer. Initial rate studies, upon varying systematically the concentrations of p-mercaptobenzoate, NADPH, and oxygen showed that the enzyme interacted with the substrates in the same manner as it does with p-hydroxybenzoate in place of the mercaptan. That is, a ternary complex is formed between enzyme, mercaptobenzoate, and NDAPH, followed by reaction and release of NADP+. Then a second ternary complex is formed between enzyme, mercaptobenzoate, and oxygen followed by reaction, liberation of product, and return to the resting state of the enzyme. Rapid reaction studies showed that the first half-reaction was analagous to that with the natural substrate. The enzyme-flavin is reduced to the 1,5-dihydroflavin by NADPH, and the rate of reaction is dramatically enhanced in the presence of mercaptobenzoate. The rate enhancement with this enzyme correlates well with the presence of a dianion form of the substrate on the enzyme. Examination of the second half-reaction showed that the reduced flavin on the enzyme formed transient intermediates upon reaction with oxygen, which were analogous to the intermediates in reactions where the enzyme forms an hydroxylated product. The oxidation of p-mercaptobenzoate by H2O2 in free solution resulted in the same disulfide as formed in the enzymatic reaction, only orders of magnitude slower. A sulfenic acid was probably the initial oxidation product from p-mercaptobenzoate, and this reacted very fast, and nonenzymatically, with mercaptobenzoate to form the disulfide and H20. The significance of the enzyme reaction with oxygen when complexed with p-mercaptobenzoate is discussed in relation to the mechanism of hydroxylation.
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PMID:Catalytic mechanism of p-hydroxybenzoate hydroxylase with p-mercaptobenzoate as substrate. 82 28

The flavoprotein p-hydroxybenzoate hydroxylase has been studied extensively by biochemical techniques by others and in our laboratory by X-ray crystallography. As a result of the latter investigations, well-refined crystal structures are known of the enzyme complexed (i) with its substrate p-hydroxybenzoate and (ii) with its reaction product 3,4-dihydroxybenzoate and (iii) the enzyme with reduced FAD. Knowledge of these structures and the availability of the three-dimensional structure of a model compound for the reactive flavin 4a-hydroperoxide intermediate has allowed a detailed analysis of the reaction with oxygen. In the model of this reaction intermediate, fitted to the active site of p-hydroxybenzoate hydroxylase, all possible positions of the distal oxygen were surveyed by rotating this oxygen about the single bond between the C4a and the proximal oxygen. It was found that the distal oxygen is free to sweep an arc of about 180 degrees in the active site. The flavin 4a-peroxide anion, which is formed after reaction of molecular oxygen with reduced FAD, might accept a proton from an active-site water molecule or from the hydroxyl group of the substrate. The position of the oxygen to be transferred with respect to the substrate appears to be almost ideal for nucleophilic attack of the substrate onto this oxygen. The oxygen is situated above the 3-position of the substrate where the substitution takes place, at an angle of about 60 degrees with the aromatic plane, allowing strong interactions with the pi electrons of the substrate. Polarization of the peroxide oxygen-oxygen bond by the enzyme may enhance the reactivity of flavin 4a-peroxide.
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PMID:Analysis of the active site of the flavoprotein p-hydroxybenzoate hydroxylase and some ideas with respect to its reaction mechanism. 233 81

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.
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PMID:Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin. 775 82

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45 degrees C. Chloride ions were inhibitory, and competitive with respect to NADH. 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-hydroxybenzoate, 2-chloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed. The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.
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PMID:4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3. Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity. 870 56

The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enzyme first catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilizing one atom of oxygen derived from water, and yielding one molecule of reduced FAD. The reducing electron equivalents are then passed one at a time from the flavin cofactor to the heme cofactor by intramolecular electron transfer, and subsequently to cytochrome oxidase within the periplasmic membrane via one or more soluble electron carrier proteins. The product, p-hydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzaldehyde. The fully refined X-ray crystal structure of PCMH in the native state has been obtained at 2. 5 A resolution on the basis of the gene sequence. The structure of the enzyme-substrate complex has also been refined, at 2.75 A resolution, and reveals significant conformational changes in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route for substrate access to the site has been identified and is shown to be governed by a swinging-gate mechanism. Two possible proton transfer pathways, that may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydrogen bonding interactions between the flavoprotein and cytochrome subunits that stabilize the intramolecular complex and may contribute to the electron transfer process have been identified.
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PMID:Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism. 1062 31

The study of redox-active systems often requires the maintenance of anaerobic conditions. The glucose oxidase system has often been used to maintain anaerobic conditions, but it has some drawbacks, such as the production of H(2)O(2) and limitations on stability. Protocatechuate dioxygenase from Burkholderia cepacia and the substrate, protocatechuate, constitute an alternate effective oxygen-scrubbing system that can be used in a wide variety of biochemical experiments. We have shown its suitability for maintaining rigorous anaerobic environments in solutions of pH 6-9, at temperatures from 4 to 35 degrees C, and for periods of time up to 15 months. The enzyme system was shown to be stable under these conditions and effective for maintaining anaerobic conditions in titrations of FAD. It is also suitable for scrubbing various types of apparatus such as stopped-flow instruments for anaerobic experiments.
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PMID:The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments. 1106 39

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl- and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.
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PMID:Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains. 1156 60

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, then oxidation of reduced FAD by oxygen to form a hydroperoxide, which oxygenates p-hydroxybenzoate to form 3,4-dihydroxybenzoate. These diverse reactions all occur within a single polypeptide and are achieved through conformational rearrangements of the isoalloxazine ring and protein residues within the protein structure. In this review, we examine the complex dynamic behavior of the protein that enables regulated fast and specific catalysis to occur. Original research papers (principally from the past 15 years) provide the information that is used to develop a comprehensive overview of the catalytic process. Much of this information has come from detailed analysis of many specific mutants of the enzyme using rapid reaction technology, biophysical measurements, and high-resolution structures obtained by X-ray crystallography. We describe how three conformations of the enzyme provide a foundation for the catalytic cycle. One conformation has a closed active site for the conduct of the oxygen reactions, which must occur in the absence of solvent. The second conformation has a partly open active site for exchange of substrate and product, and the third conformation has a closed protein structure with the isoalloxazine ring rotated out to the surface for reaction with NADPH, which binds in a surface cleft. A fundamental feature of the enzyme is a H-bond network that connects the phenolic group of the substrate in the buried active site to the surface of the protein. This network serves to protonate and deprotonate the substrate and product in the active site to promote catalysis and regulate the coordination of conformational states for efficient catalysis.
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PMID:Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase. 1558 85

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis.
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PMID:Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase. 1592 24

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.
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PMID:Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase. 1620 56


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