Balance between photoreduction efficiency, cofactor affinity, and allosteric coupling of halogenase flavoenzymes.

Abstract

Flavin-dependent halogenases (FDHs) are promising candidates for the sustainable production of halogenated organic molecules by biocatalysis. FDHs require only oxygen, halide and a fully reduced flavin adenine dinucleotide (FADH^-) cofactor to generate the reactive HOX that diffuses 10 Å to the substrate binding pocket and enables regioselective oxidative halogenation. A key challenge for the application of FDHs is the regeneration of the FADH^-. In vitro, FADH^- can be regenerated by photoreduction of the oxidized FAD inside the protein using blue light, turning the halogenase into an inefficient artificial photoenzyme. We aimed to improve the photochemical properties of the tryptophan 5-halogenase PyrH from Streptomyces rugosporus by structure-guided mutagenesis. W279 and W281 of the conserved WxWxIP-motif close to FAD were exchanged against phenylalanine. Time-resolved UV-vis spectroscopy showed that the W281F exchange indeed increased the quantum yield of the one- and two-electron reduction, respectively. The cofactor binding affinity decreased slightly with dissociation constants rising from 31 to 74 μM, as examined by fluorescence anisotropy. FTIR difference spectroscopy demonstrated that the allosteric coupling between the FAD and substrate binding sites was mostly preserved. In contrast, the double mutant did not improve the yield further, while negatively affecting binding affinity and structural coupling. The distal W279F exchange was less effective in all parameters. Photoreductions were additionally delayed by a reversible inactive conformation. We conclude that there is a delicate balance to be considered for screening of FDHs for biocatalysis. Variant PyrH-W281F was found to be the most promising candidate for the application as artificial photoenzyme.