Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize a histidine-bound vanadate cofactor and hydrogen peroxide to oxidize a halide to hypohalous acid. About 10 years ago, Streptomycete homologs were discovered to be involved in the site-selective and substrate-specific halogenation of meroterpenoid natural products (NPs). Since then, site-selective VHPOs have remained under-explored despite their broad species distribution and variable genomic contexts. Although only a few closely related VHPOs have been characterized, a diverse range of chemical transformations have been observed. Recently, the first VHPO (VHPO-HZ11) from a non-Streptomyces species was characterized to site-specifically brominate alkyl quinolones (AQs). AQs are well known for their role in Pseudomonas aeruginosa pathogenesis, however they also play an important role in marine interspecies communication. AQ bromination results in reduced toxicity to the VHPO-containing organism and increased AQ toxicity to other bacteria. We propose that VHPO-HZ11 and its homologs are an excellent sub-family to develop a model for the mechanism of selective VHPO substrate modification and for identifying the larger role these enzymes play in the microbial environment. We have identified additional VHPO-HZ11 homologs from a wide range of bacterial taxa including: Actinobacteria, Acidobacteria, Chloroflexi, and Proteobacteria. There were no conserved genomic contexts for these VHPOs and a general lack of AQ-producing enzymes in the organisms, implying a role outside of endogenous NP biosynthesis. Heterologous expression and in vitro interrogation of these VHPO homologs with several AQ substrates has shown a conservation of halogenation activity across species. Initial kinetic studies suggest that the AQ-modifying VHPOs are more catalytically robust compared to their meroterpenoid biosynthetic counterparts. We are currently initiating site-directed mutagenesis to understand the sequence requirements for site-selective halogenation and co-culturing experiments to understand the role that VHPO-mediated AQ modification plays in microbial communication.

Investigating the role of alkyl quinolone modifying vanadium-dependent haloperoxidases in microbial community modulation

ACS Spring 2022

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