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)

Consensus amino acid sequences of FADH(2)-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a beta-alpha-beta fold present in FAD-binding sites of certain monooxygenases. When used to probe Southern blots of restriction-enzyme-digested DNA, the [alpha-(32)P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes.
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PMID:Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230. 1208 Apr 19

Chlorination of natural products is often required for their biological activity; notable examples include vancomycin, the last-ditch antibiotic. It is now known that many chlorinated natural products are made not by haloperoxidases, but by FADH2-dependent halogenases. The mechanism of the flavin-containing enzymes is obscure and there are no structural data. Here, crystals of PrnA (tryptophan 7-halogenase), an enzyme that regioselectively chlorinates tryptophan, cocrystallized with tryptophan and FAD are reported. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 67.8, c = 276.9 A. A data set to 1.8 A with 93% completeness and an Rmerge of 7.1% has been collected from a single flash-cooled crystal. A method for incorporating selenomethionine in a Pseudomonas fluorescens expression system also is reported.
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PMID:Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens. 1527 70

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k(cat) of 1.4 min(-1) was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.
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PMID:Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis. 1574 14

The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH2. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCl intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.
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PMID:Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis. 1616 66

The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH(2) (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH(2) and O(2) in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)-OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)-OH intermediate. The order of addition of FADH(2) and O(2) was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s(-)(1), quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s(-)(1) at 25 degrees C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH(2), chloride ion, and O(2).
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PMID:Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH. 1678 43

The lichen cyanobacterial symbiont Nostoc sp. ATCC 53789 and its close relative Nostoc sp. GSV 224 are prolific producers of natural products, generating >25 derivatives of the cryptophycin class of secondary metabolites. Cryptophycin 1, the prototypic member of the class, is a potent tubulin-depolymerizing agent, and several semisynthetic derivatives are being developed as anticancer therapeutics. Here we provide a detailed characterization of the cryptophycin metabolic pathway by stable-isotope labeling experiments and through cloning, sequencing, and annotating the cryptophycin biosynthetic gene cluster. A comparative secondary metabolomic analysis based on polyketide (PK)/non-ribosomal peptide gene clusters from the phylogenetically related, non-cryptophycin producing cycad symbiont, Nostoc punctiforme ATCC 29133, was used to identify the cryptophycin biosynthetic genes that encompass approximately 40 kb within the lichen symbiont Nostoc sp. ATCC 53789 genome. The pathway encodes a collinear set of enzymes, including three modular PK synthases, two non-ribosomal peptide synthetase modules, and an integrated adenylation/ketoreductase didomain for elaboration of the leucic acid subunit. In addition, genes encoding key tailoring steps, including a FAD-dependent halogenase and CYP450 epoxidase, were identified. The inherent flexibility of the cryptophycin biosynthetic enzymes was harnessed to generate a suite of new analogues by altering the pool of PK starter units and selected amino acid extender groups. Characterization of the cryptophycin CYP450 enabled development of the first stereospecific synthesis of cryptophycin 2, through a tandem chemoenzymatic synthesis from the natural seco-cryptophycin 4 chain elongation intermediate.
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PMID:Biosynthetic characterization and chemoenzymatic assembly of the cryptophycins. Potent anticancer agents from cyanobionts. 1724 Sep 71

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.
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PMID:Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases. 1726 Sep 57

The crystal structure of the FAD-dependent chondrochloren halogenase CndH has been established at 2.1 A resolution. The enzyme contains the characteristic FAD-binding scaffold of the glutathione reductase superfamily. Except for its C-terminal domain, the chainfold of CndH is virtually identical with those of FAD-dependent aromatic hydroxylases. When compared to the structurally known FAD-dependent halogenases PrnA and RebH, CndH lacks a 45 residue segment near position 100 and deviates in the C-terminal domain. Both variations are near the active center and appear to reflect substrate differences. Whereas PrnA and RebH modify free tryptophan, CndH halogenates the tyrosyl group of a chondrochloren precursor that is most likely bound to a carrier protein. In contrast to PrnA and RebH, which enclose their small substrate completely, CndH has a large non-polar surface patch that may accommodate the putative carrier. Apart from the substrate binding site, the active center of CndH corresponds to those of PrnA and RebH. At the halogenation site, CndH has the characteristic lysine (Lys76) but lacks the required base Glu346 (PrnA). This base may be supplied by a residue of its C-terminal domain or by the carrier. These differences were corroborated by an overall sequence comparison between the known FAD-dependent halogenases, which revealed a split into a PrnA-RebH group and a CndH group. The two functionally established members of the CndH group use carrier-bound substrates, whereas three members of PrnA-RebH group are known to accept a free amino acid. Given the structural and functional distinction, we classify CndH as a new variant B of the FAD-dependent halogenases, adding a new feature to the structurally established variant A enzymes PrnA and RebH.
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PMID:Structure and action of the myxobacterial chondrochloren halogenase CndH: a new variant of FAD-dependent halogenases. 1900 Jun 96

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an (S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme.
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PMID:Characterization of SgcE6, the flavin reductase component supporting FAD-dependent halogenation and hydroxylation in the biosynthesis of the enediyne antitumor antibiotic C-1027. 1981 65

The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085. Sequence analysis of a 110,633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaG1 and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P450s (TiaP1 and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis.
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PMID:Characterization of tiacumicin B biosynthetic gene cluster affording diversified tiacumicin analogues and revealing a tailoring dihalogenase. 2118 5


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