153-97-9Relevant articles and documents
Tandem action of the O2- and FADH2-dependent halogenases KtzQ and KtzR produce 6,7-dichlorotryptophan for kutzneride assembly
Heemstra Jr., John R.,Walsh, Christopher T.
, p. 14024 - 14025 (2008)
Kutznerides are actinomycete-derived antifungal nonribosomal hexadepsipeptides which are assembled from five unsual nonproteinogenic amino acids and one hydroxy acid. Conserved in all structurally characterized kutznerides is a dichlorinated tricyclic hexahydropyrroloindole postulated to be derived from 6,7-dichlorotryptophan. In this Communication, we identify KtzQ and KtzR as tandem acting FADH2-dependent halogenases that work sequentially on free l-tryptophan to generate 6,7-dichloro-l-tryptophan. Kinetic characterization of these two enzymes has shown that KtzQ (along with the flavinreductase KtzS) acts first to chlorinate at the 7-position of l-tryptophan. KtzR, with a ～120 fold preference for 7-chloro-l-tryptophan over l-tryptophan, then installs the second chlorine at the 6-position of 7-chloro-l-tryptophan to generate 6,7-dichloro-l-tryptophan. These findings provide further insights into the enzymatic logic of carbon-chloride bond formation during the biosynthesis of halogenated secondary metabolites. Copyright
Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes
Andorfer, Mary C.,Belsare, Ketaki D.,Girlich, Anna M.,Lewis, Jared C.
, p. 2099 - 2103 (2017)
The remarkable site selectivity and broad substrate scope of flavin-dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required. Herein, we report functional synthetic, fused FDH-FR proteins containing various FDHs and FRs joined by different linkers. We show that FDH-FR fusion proteins can increase product titers compared to the individual components for in vivo biocatalysis in Escherichia coli.
FADH2-dependence of tryptophan 7-halogenase
Unversucht, Susanne,Hollmann, Frank,Schmid, Andreas,Van Pee, Karl-Heinz
, p. 1163 - 1167 (2005)
Tryptophan 7-halogenase (Trp 7-hal) catalyses the regioselective chlorination and bromination of tryptophan. For halogenating activity, Trp 7-hal requires FADH2 produced from FAD and NADH by a flavin reductase, halide ions (chloride or bromide), molecular oxygen and tryptophan as the organic substrate. Investigations of the flavin dependence showed that purified Trp 7-hal itself does not contain flavin. Keeping the Trp 7-hal separated from the flavin reductase during the reaction revealed that Trp 7-hal can use diffusible FADH2 produced by a flavin reductase showing that direct contact between the halogen ase and the flavin reductase is not absolutely necessary. Thus, the reaction also proceeds when chemically reduced flavin is used. For the catalytic regeneration of FADH2, the organometallic complex (pentamethylcyclopentadienyl)rhodium-bipyridine {[Cp*Rh(bpy) (H2O)]2+} can be employed as the redox catalyst with formate as the electron donor. With this chemoenzymatic system about 85% yields of the product formed by the two-component enzyme system consisting of Trp 7-hal and a flavin reductase were obtained.
Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal
Moritzer, Ann-Christin,Minges, Hannah,Prior, Tina,Frese, Marcel,Sewald, Norbert,Niemann, Hartmut H.
, p. 2529 - 2542 (2019)
Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH2, oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate tryptophan’s indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 ? resolution. The structure revealed that the C6 of tryptophan is positioned next to the -amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electro-philic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of L-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.
Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation
Ismail, Mohamed,Schroeder, Lea,Frese, Marcel,Kottke, Tilman,Hollmann, Frank,Paul, Caroline E.,Sewald, Norbert
, p. 1389 - 1395 (2019/02/10)
Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogena
