Reinigung und teilweise Charakterisierung der Tryptophan-7-Halogenase (PrnA) ausPseudomonas fluorescens

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

doi:10.1002/1521-3757(20000703)112:13<2380::aid-ange2380>3.0.co;2-2

Cited by 29 publications

(3 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The native and mutant proteins were produced in Pseudomonas fluorescens BL915 Δ ORF 1–4 with and without a His tag. His-tagged enzymes were purified using a Ni-chelating sepharose FF column and non-His-tagged enzymes were partially purified using a Q-sepharose FF column (Amersham Biosciences) as described by Keller et al5 The activity of PrnA was determined according to the method described previously 5, 7. The reaction mixture was composed of 55 μL PrnA-containing protein solution, 10.8 mU FAD reductase, 10 μm FAD, 2.4 mm NADH, 12.5 mm MgCl2, 100 U catalase, and 0.6 mm tryptophan in a total volume of 200 μL in 10 mm potassium phosphate buffer, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

“…Although in a number of cases, a flavin reductase gene is present in the biosynthetic gene cluster of the halometabolites, it is unclear whether the corresponding flavin reductases interact directly with the halogenases. At least in a number of cases, flavin reductases from different bacterial strains can be used in combination with halogenases 1, 2–5. For the tryptophan 7-halogenase PrnA from Pseudomonas fluorescens BL915 which catalyzes the first step in pyrrolnitrin biosynthesis6 it could be shown that even chemically reduced FAD is used by the halogenase in the halogenation reaction 7.…”

mentioning

confidence: 99%

See 1 more Smart Citation

New Insights into the Mechanism of Enzymatic Chlorination of Tryptophan

et al. 2008

View full text Add to dashboard Cite

Flavin-dependent halogenases have been shown to play a major role in biological halogenation reactions. For halogenating activity, flavin-dependent halogenases require reduced FAD, which is formed from FAD and NADH by a second enzyme, a flavin reductase. Although in a number of cases, a flavin reductase gene is present in the biosynthetic gene cluster of the halometabolites, it is unclear whether the corresponding flavin reductases interact directly with the halogenases. At least in a number of cases, flavin reductases from different bacterial strains can be used in combination with halogenases.1, 2-5 For the tryptophan 7-halogenase PrnA from Pseudomonas fluorescens BL915 which catalyzes the first step in pyrrolnitrin biosynthesis6 it could be shown that even chemically reduced FAD is used by the halogenase in the halogenation reaction.7 Based on the threedimensional structure of PrnA, it was postulated that flavin hydroperoxide is formed by the reaction of halogenase-bound reduced flavin with oxygen. This flavin hydroperoxide then reacts with chloride ion leading to the formation of hypochlorous acid, which is then guided along a tunnel about 10 Å long towards the substrate tryptophan (Figure 1). A lysine residue (K79) was suggested to hydrogen bond with hypochlorous acid and thus position it to react with tryptophan.8 Yeh et al. demonstrated chloramine formation by the reaction of HOCl with the ε-amino group of lysine,9 suggesting that chloramine rather than HOCl is the active agent. The importance of K79 is undisputed; exchange of K79 against an alanine residue leads to total loss of halogenating activity as demonstrated for PrnA8 and for the tryptophan 7-halogenase RebH from rebeccamycin biosynthesis.9 However, other factors must also be at work, since chlorination of tryptophan cannot be accomplished by chloramine (or HOCl) in solution.10, 11 Chloramine is a weaker halogenating agent than HOCl,12 and according to quantum mechanical calculations, N-chloramine formation reduces the electrophilicity ofthe chlorine species; in other words, the charge Q(Cl) is reduced to −0.07 compared to Q(Cl)=+0.017 in free HOCl.In the active site, glutamate 346 (E346) is positioned across the tunnel from K79, and the positioning of the substrate tryptophan is supported by a hydrogen bond between the NH group of the indole ring and the peptide bond oxygen between E346 and serine 347 (S347) (Figure 1). Evidence that E346 could be involved in the catalytic cycle was the observation that E346Q is two orders of magnitude less active.8 Whereas K79 is absolutely conserved in all the flavin-dependent halogenases known so far, E346 and S347 are conserved only in flavin-dependent tryptophan halogenases and they are not present in halogenases acting on substrates with a phenol or pyrrole ring. Dong et al. suggested that E346 is required for the ‡ Equal contribution

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

New Insights into the Mechanism of Enzymatic Chlorination of Tryptophan

et al. 2008

View full text Add to dashboard Cite

show abstract

“…For the application of this enzyme group, it is important to keep in mind that they need at least a two-component electron transport chain and therefore require a suitable flavin reductase [90,91,92]. In addition to the reductases that naturally belong to the biosynthesis clusters e.g., PrnF [93], applications with other reductases such as SsuE [91,94,95] or Fre [96,97] from E. coli have also been reported. To avoid the necessity of a second enzyme—the flavin reductase—or even a third enzyme for cofactor recycling, photochemical approaches are in the focus of current research in this field as well [98].…”

Section: Metal-free Haloperoxidases/perhydrolasesmentioning

confidence: 99%

Halogenating Enzymes for Active Agent Synthesis: First Steps Are Done and Many Have to Follow

et al. 2019

View full text Add to dashboard Cite

Halogens can be very important for active agents as vital parts of their binding mode, on the one hand, but are on the other hand instrumental in the synthesis of most active agents. However, the primary halogenating compound is molecular chlorine which has two major drawbacks, high energy consumption and hazardous handling. Nature bypassed molecular halogens and evolved at least six halogenating enzymes: Three kind of haloperoxidases, flavin-dependent halogenases as well as α-ketoglutarate and S-adenosylmethionine (SAM)-dependent halogenases. This review shows what is known today on these enzymes in terms of biocatalytic usage. The reader may understand this review as a plea for the usage of halogenating enzymes for fine chemical syntheses, but there are many steps to take until halogenating enzymes are reliable, flexible, and sustainable catalysts for halogenation.

show abstract

Der Aufbau von Vancomycin: so macht es die Natur

Hubbard

Walsh

2003

Angewandte Chemie

View full text Add to dashboard Cite

Antibiotika sind wertvolle Therapeutika im Kampf gegen Infektionen, die durch pathogene Bakterien verursacht werden. Vancomycin ist eine der letzten wirksamen “Waffen” bei lebensbedrohlichen Infektionen durch Gram‐positive Bakterien. Die Regeln, nach denen die Natur die Glycopeptid‐ (Vancomycin) und Lipoglycopeptid‐Antibiotika (Teicoplanin) aufbaut, werden zunehmend geklärt und abgesichert: Zuerst werden Aminosäuren synthetisiert, diese anschließend zusammengebaut und vernetzt. Damit öffnen sich Möglichkeiten, die Synthesestrategien umzuprogrammieren auf der Ebene veränderter Monomere, durch Verschiebungen beim Peptidaufbau und durch verschiedene Enzyme zur Nachbearbeitung des Aglycons.

show abstract

Reinigung und teilweise Charakterisierung der Tryptophan-7-Halogenase (PrnA) ausPseudomonas fluorescens

Cited by 29 publications

References 20 publications

New Insights into the Mechanism of Enzymatic Chlorination of Tryptophan

New Insights into the Mechanism of Enzymatic Chlorination of Tryptophan

Halogenating Enzymes for Active Agent Synthesis: First Steps Are Done and Many Have to Follow

Der Aufbau von Vancomycin: so macht es die Natur

Contact Info

Product

Resources

About