• 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • br Halogenases Enzymatic C H


    Halogenases Enzymatic C–H activation leading to halogenation is another emerging area in biocatalysis [55,56]. Incorporation of halogen atoms during medicinal chemistry eff ;orts is a well-established practice, presenting an effective means to control the molecule’s bioactivity and physicochemical properties [57]. Additionally, C–X motifs offer chemical handles for further transformations via metal-catalyzed cross-coupling and nucleophilic substitutions [58]. The majority of halogenases utilize an oxidative strategy and can be divided into four subclasses: heme-dependent halogenases, vanadium-dependent halogenases, flavin-dependent halogenases, and non-heme iron-dependent halogenases [56]. While the two former subclasses generally lack selectivity due to the unspecific release of hypohalous Berberine Sulfate synthesis as the halogenating agent, flavin-dependent and non-heme iron-dependent enzymes act via a defined, protein-bound reactive species which enables regioselective and stereoselective halogen transfer [59] (Figures 1b and 2 ). Flavin-containing halogenases, particularly tryptophan halogenases, have been subjected to extensive engineering [,61] (Figure 2). Andorfer et al. reported engineered variants of 7-tryptophane halogenase RebH which are capable of chlorinating indole derivatives ortho, meta, and para to the indole nitrogen []. Starting from a thermostable RebH variant [62], mutant libraries were generated by the combination of epPCR and site directed mutagenesis and then screened via mass spectrometry for the regioselective chlorination of a deuterium-substituted probe substrate. A total of six rounds of evolution afforded two variants capable of chlorinating the 5-position or 6-position of the indole substrate with >90% regioselectivity and reasonable conversion rates showing that directed evolution provides a means to systematically tune the enzyme active site to functionalize different C–H bonds on a given substrate (Figure 2c). Using the same starting variant, the enzyme was also tailored to allow the late-stage halogenation of a number of large (ca. 400 Da) bioactive substrates [61] such as Carvedilol and Yohimbine with high regioselectivity (Figure 2d). Another class of halogenating enzymes are non-heme iron α-KG-dependent halogenases, close homologues of the α-KGDs. As in α-KG hydroxylases a high-valent Fe(IV)-oxo species abstracts a hydrogen atom from an unactivated sp3 carbon center of the substrate molecule before a halogen radical rebinds [63]. This difference in reactivity (i.e. halogenation versus hydroxylation) is likely due to the conserved metal binding motifs in the two enzymes: His-X--Xn-His in hydroxylases versus His-X--Xn-His in halogenases. The latter provide an open coordination site for a halogen atom (Figure 1b). The majority of non-heme iron α-KG-dependent halogenases acts on substrates tethered to acyl or peptidyl carrier proteins limiting applicability [63]. However, the recent discovery of WelO5 and the more promiscuous AmbO5 from cyanobacterium Fischerella ambigua, which are both capable of halogenating unbound molecules, opens up the possibility to functionalize free-standing substrates [64, 65, 66] (Figure 1b). Interestingly, the reason for AmbO5’s ability to derivatize a broader set of molecules than its close homolog WelO5 was identified to reside in its C-terminal sequence motif. Transfer of this subunit to WelO5 resulted in a functional variant with an expanded substrate scope identical to AmbO5 [67]. Overall, these results indicate the evolvable nature of halogenases, which may be exploited in their further utilization for the conversion of both natural and non-natural substrate molecules. Although the number of α-KG-dependent halogenases reported to date is significantly lower than for the related hydroxylases, structure-guided reprogramming of hydroxylases to halogenases could lead to more enzymes available for biocatalytic halogenation applications []. Mitchell et al. recently reported that replacement of the carboxylate ligand with glycine in an α-KG hydroxylase SadA resulted in conversion to a halogenase with residual hydroxylase activity []. The variant with the single mutation was shown to mono-chlorinate or brominate an amino acid derivative with high regioselectivity and stereoselectivity (Figure 1b).