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

3-Hydroxyphenylacetate 6-hydroxylase was purified 70-fold from a Flavobacterium sp. grown upon phenylacetic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme. SDS/PAGE of purified enzyme reveals a single band with a minimum molecular mass of 63 kDa. Analytical gel-filtration, sedimentation-equilibrium and sedimentation-velocity experiments indicate that the purified enzyme exists in solution mainly as a dimer, containing 1 molecule non-covalently bound FAD/subunit. 3-Hydroxyphenylacetate 6-hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3-hydroxyphenylacetate is hydroxylated efficiently, yielding 2,5-dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4-dihydroxyphenylacetate and 4-hydroxyphenylacetate are partially hydroxylated, exclusively at the 6' (2') position. The physiological product 2,5-dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3-hydroxyphenylacetate 6-hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate. In the native state of enzyme, two sulfhydryl groups are accessible to 5,5'-dithiobis(2-nitrobenzoate). Titration with stoichiometric amounts of either 5,5'-dithiobis(2-nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3-hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol. The N-terminal amino acid sequence of the enzyme lacks the consensus sequence GXGXXG, found at the N-termini of all flavin-dependent external monooxygenases sequenced so far. The amino acid composition of 3-hydroxyphenylacetate 6-hydroxylase is also presented.
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PMID:Purification and characterisation of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacterium species. 193 54

Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The enzyme has a molecular weight of 47 955 +/- 39, as determined by MALDI-TOF mass spectrometry; under nondenaturing conditions, the aggregation state of the enzyme is best described by a tetramer-dimer self-associating model, with an association constant of (8.5 +/- 4.4) x 10(6) M-1 (pH 7.0 and 4 degreesC). The amino acid composition and the N-terminal amino acid sequence do not match any known protein or open reading frame. The inactive 5-nitrobutyl-1,5-dihydroflavin found in the enzyme as purified was converted to FAD, allowing characterization of the active FAD-containing enzyme. With nitroethane as substrate, the Vmax and Km values are 655 +/- 45 min-1 and 2.9 +/- 0.5 mM at pH 8.0 and 30 degreesC, respectively. One mole of FAD per mole of monomer enzyme is required for catalysis. No activity can be detected with amino acids or alpha-hydroxy acids as substrates. Reversible removal of the FAD cofactor yields inactive enzyme. The properties of the FAD cofactor in nitroalkane oxidase are within the range described for other oxidases. The UV-visible absorbance spectrum of the active enzyme shows maxima at 446, 384, and 274 nm; the extinction coefficient at 446 nm is 11.7 mM-1 cm-1. The neutral form of the flavin semiquinone, with maxima at 536 and 342 nm, is kinetically stabilized. The UV-visible absorbance spectrum of the reduced enzyme is typical of the anionic form of a flavin, with a peak centered at 335 nm. The affinity of the enzyme for sulfite is low (Kd value of 13.8 +/- 0.9 mM at pH 7.0 and 25 degreesC); this result, along with the stabilization of the neutral flavin semiquinone, suggests the presence of a weak positive charge near the N(1)-C(2)=O of FAD. The reduction potential of the enzyme is -367 mV. Benzoate and phenylacetic acid are competitive inhibitors, with Kis values of 5.1 +/- 0.6 and 13.1 +/- 2.3 mM, respectively. Binding of benzoate to nitroalkane oxidase results in spectral changes similar to those observed with d-amino acid oxidase. The absorbance spectrum of the flavin bound to nitroalkane oxidase is pH-dependent, with a pKa value of 8.4.
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PMID:Biochemical and physical characterization of the active FAD-containing form of nitroalkane oxidase from Fusarium oxysporum. 955 55

Annotation of genome of Streptomyces peucetius revealed a putative phenylacetic acid degradation NADH oxidoreductase. RT-PCR analysis of the gene readily showed notable transcription in its native state. The transcription level of paaE when the host is grown on phenylacetic acid showed increased transcription. paaE was cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in Escherichia coli BL21(DE3) and purified by immobilized metal affinity chromatography using His-tag. The flavin released from heat-denatured PaaE was identical to that of authentic FAD in HPLC analysis. The purified protein efficiently reduced p-nitroblue tetrazolium (an electron acceptor) in presence of NADH. Cell growth analysis of S. peucetius in phenylacetic acid evidently revealed its involvement in degradation of phenylacetic acid - a key environmental pollutant.
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PMID:Identification and characterization of a NADH oxidoreductase involved in phenylacetic acid degradation pathway from Streptomyces peucetius. 2011 24

Petroleum is the major energy matrix in the world whose refining generates chemical byproducts that may damage the environment. Among such waste, polycyclic aromatic hydrocarbons (PAH) are considered persistent pollutants. Sixteen of these are considered priority for remediation, and among them is benzo(a)pyrene. Amid remediation techniques, bioremediation stands out. The genus Burkholderia is amongst the microorganisms known for being capable of degrading persistent compounds; its strains are used as models to study such ability. High-throughput sequencing allows researchers to reach a wider knowledge about biodegradation by bacteria. Using transcripts and mRNA analysis, the genomic regions involved in this aptitude can be detected. To unravel these processes, we used the model B. vietnamiensis strain G4 in two experimental groups: one was exposed to benzo(a)pyrene and the other one (control) was not. Six transcriptomes were generated from each group aiming to compare gene expression and infer which genes are involved in degradation pathways. One hundred fifty-six genes were differentially expressed in the benzo(a)pyrene exposed group, from which 33% are involved in catalytic activity. Among these, the most significant genomic regions were phenylacetic acid degradation protein paaN, involved in the degradation of organic compounds to obtain energy; oxidoreductase FAD-binding subunit, related to the regulation of electrons within groups of dioxygenase enzymes with potential to cleave benzene rings; and dehydrogenase, described as accountable for phenol degradation. These data provide the basis for understanding the bioremediation of benzo(a)pyrene and the possible applications of this strain in polluted environments.
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PMID:Differential Expression and PAH Degradation: What Burkholderia vietnamiensis G4 Can Tell Us? 3290 29