Purification and Partial Characterization of Tryptophan 7-Halogenase (PrnA) fromPseudomonas fluorescens

Keller, Sascha; Wage, Tobias; Hohaus, Kathrin; Hölzer, Manuela; Eichhorn, Eric; Pée, Karl‐Heinz van

doi:10.1002/1521-3773(20000703)39:13<2300::aid-anie2300>3.0.co;2-i

Cited by 181 publications

(177 citation statements)

References 11 publications

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“…In addition to the requirement for a flavin reductase to provide reduced flavin cofactor, previous studies of FADH 2 -dependent halogenases indicated that the reaction also depended on O 2 (9). For that reason, RebF͞RebH reactions were prepared anaerobically, and O 2 was subsequently introduced to initiate the reaction.…”

Section: Formation Of 7-chlorotryptophan By Rebh In Presence Ofmentioning

confidence: 99%

“…Despite the many representatives of this halogenase class and its role in the biosynthesis of medicinally important halogenated aromatic natural products, only a few of these catalysts have been characterized (9)(10)(11). PrnA, the Trp-7-halogenase involved in the formation of pyrrolnitrin, is foremost among FADH 2 -dependent halogenases that have been described (9,10).…”

mentioning

confidence: 99%

“…PrnA, the Trp-7-halogenase involved in the formation of pyrrolnitrin, is foremost among FADH 2 -dependent halogenases that have been described (9,10). In these studies, van Pée and coworkers (9,10) established the requirement for a separate flavin reductase to provide reduced FADH 2 for the halogenase, in analogy with two-component flavin monooxygenase systems (12)(13)(14). However, low in vitro activity of this system has precluded more detailed studies, so even a parameter as fundamental as catalytic efficiency has yet to be reported for a FADH 2 -dependent halogenase, and little is known about the mechanism of halogenation by these enzymes.…”

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confidence: 99%

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Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Yeh

Garneau²,

Walsh³

2005

Proc. Natl. Acad. Sci. U.S.A.

256

278

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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 FADH 2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O 2, 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 FADH 2 for the halogenase. Maximal rates were achieved at a RebF͞RebH ratio of 3:1. In air-saturated solutions, a k cat of 1.4 min ؊1 was observed for the RebF͞RebH system but increased at least 10-fold in low-pO 2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF͞RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.enzymology ͉ natural product biosynthesis ͉ flavoenzyme

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Section: Formation Of 7-chlorotryptophan By Rebh In Presence Ofmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Yeh

Garneau²,

Walsh³

2005

Proc. Natl. Acad. Sci. U.S.A.

256

278

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“…In FAD-dependent hydroxylations the flavin is reduced by NAD(P)H and reacts with oxygen to form the 4(a)-flavin hydroperoxide; the hydroperoxide oxidizes the substrate to an epoxide, which is then opened by nucleophilic attack from a hydroxyl ion (Entsch et al, 1976;Gatti et al, 1994). A similar mechanism proposed for halogenasecatalysed reactions (Hohaus et al, 1997;Keller et al, 2000) is supported by recent evidence (van Pée, 2001) that the halogenase gene product requires FAD, NADPH, reductase activity and oxygen to function as a halogenating system. The epoxide generated by the flavin hydroperoxide undergoes nucleophilic attack by a halide ion, and specific removal of water gives the halogenated product.…”

Section: Function Of Cmlsmentioning

confidence: 99%

“…Nevertheless, experiments with isotopically labelled potential precursors such as acetate, malonate and acetoacetate (Simonsen et al, 1978;Groß et al, 2002) have not firmly identified the substrate that, upon chlorination, provides the activated dichloroacetyl intermediate. Recently new avenues for exploring the mechanisms that introduce halogens into natural products have been opened by the discovery of FADH 2 -dependent halogenases (van Pée, 2001), and by demonstrations of their role in the biosynthesis of halogenated secondary metabolites (Hohaus et al, 1997;Kirner et al, 1998;NowakThompson et al, 1999;Keller et al, 2000;Puk et al, 2002). Halogenase genes have now been cloned from a variety of organisms: e.g.…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

2004

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Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1 H-NMR established that the principal metabolite in the disrupted strains was the a-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH 2 -dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

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Flavoenzyme-Catalyzed Oxygenations and Oxidations of Phenolic Compounds

Moonen

Fraaije

Rietjens

et al. 2002

Advanced Synthesis & Catalysis

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Flavin‐dependent monooxygenases and oxidases play an important role in the mineralization of phenolic compounds. Because of their exquisite regioselectivity and stereoselectivity, these enzymes are of interest for the biocatalytic production of fine chemicals and food ingredients. In our group, we have characterized several flavoenzymes that act on phenolic compounds, including 4‐hydroxybenzoate 3‐hydroxylase, 3‐hydroxyphenylacetate 6‐hydroxylase, 4‐hydroxybenzoate 1‐hydroxylase (decarboxylating), hydroquinone hydroxylase, 2‐hydroxybiphenyl 3‐monooxygenase, phenol hydroxylase, 4‐hydroxyacetophenone monooxygenase and vanillyl‐alcohol oxidase. The catalytic properties of these enzymes are reviewed here, together with insights obtained from site‐directed and random mutagenesis.

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Purification and Partial Characterization of Tryptophan 7-Halogenase (PrnA) fromPseudomonas fluorescens

Cited by 181 publications

References 11 publications

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

Flavoenzyme-Catalyzed Oxygenations and Oxidations of Phenolic Compounds

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