Vanadium haloperoxidases as noncanonical terpene synthases

Regioselective Halogenation of Lavanducyanin by a Site-Selective Vanadium-Dependent Chloroperoxidase

“…Purified His 6 -LvcH (2 mg/L) was obtained at sufficient purity for downstream enzymatic reactions with 1 (Figure S3). We observe a duplication in the SDS-PAGE gel for LvcH consistent with the presence (58 kDa) and absence (54 kDa) of the N-terminal His 6 -tag and signal peptide; we have observed this phenomenon with other recombinantly expressed actinobacterial VHPOs . Subsequent size exclusion chromatography purification revealed that LvcH exists as a homodimer in solution, rationalizing how the smaller construct lacking the N-terminal hexahistidine tag could be purified by using IMAC.…”

“…CNZ-289 VHPOs that catalyzed the monohalogenation of 1 under lysate conditions. Since NapH1 and NapH4 are site-selective napyradiomycin chloroperoxidases capable of nonselectively releasing HOBr, ,, the homologous 289 VH6 and 289 VH8 could serve as controls for nonselective haloperoxidase activity. 289 VH7 was omitted from this analysis since its NapH3 homologue catalyzes a thermodynamically favorable α-hydroxyketone rearrangement instead of halogenation biochemistry. , A mass ion peak for iodinated 1 was observed in each crude VHPO lysate; however, iodide can spontaneously oxidize to HOI in the presence of hydrogen peroxide and vanadium so this is not indicative of site specific halide installation. , Despite halogenating a phenazine-derived substrate, 289 VH5 displays 53–59% sequence identity with characterized meroterpenoid VHPOs (Table S6).…”

Regioselective halogenation of lavanducyanin by a site-selective vanadium-dependent chloroperoxidase

“…We observe a duplication in the SDS-PAGE gel for LvcH consistent with the presence (58 kDa) and absence (54 kDa) of the N-terminal His6-tag and signal peptide; we have observed this phenomenon with other recombinantly expressed actinobacterial VHPOs. 7 Subsequent size exclusion chromatography purification revealed that LvcH exists as a homodimer in solution, rationalizing how the smaller construct lacking the Nterminal hexahistidine tag could be purified using IMAC. A significant proportion of purified LvcH was soluble aggregate and excluded from further biochemical characterization (Figure S4).…”

“…5 However, site-selective VHPOs with specific roles in natural product biosynthesis or quorum sensor modification have been more recently characterized. [6][7][8] Despite catalyzing dramatic carbon skeleton rearrangements and the asymmetric installation of halogens, 9,10 the known pool of substrate scaffolds for these halogenases remains small. To discover novel VHPO substrates, we focused our efforts on the MAR4 clade of Streptomyces which are genomically enriched in VHPOs and abundantly produce halogenated meroterpenoid natural products, 11 including the majority of known halogenated phenazines.…”

“…CNZ-289 VHPOs catalyzed the monohalogenation of 1 under lysate conditions. Since NapH1 and NapH4 are site-selective napyradiomycin chloroperoxidases capable of non-selectively releasing HOBr, 6,7,9 the homologous 289VH6 and 289VH8 could serve as controls for non-selective haloperoxidase activity. 289VH7 was omitted from this analysis since its NapH3 homolog catalyzes a thermodynamically favorable a-hydroxyketone rearrangement instead of halogenation biochemistry.…”