UC Santa Cruz

Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize a histidine-bound vanadate cofactor and hydrogen peroxide to oxidize aqueous halides to hypohalous acid. Historically, VHPO activity has been characterized to release aqueous hypohalous acids that spontaneously halogenate electron rich molecules; this generation of reactive halogen species and the general stability of this enzyme family has led to some application in biocatalysis. Contrastingly, certain bacterial homologs have been discovered to catalyze the regio- and/or enantio- selective halogenation of meroterpenoid and alkyl quinolone (AQ) natural products. Enzymatic control over halogen installation is a powerful tool for biocatalytic and chemoenzymatic syntheses, however a lack of understanding about the mechanism of selective halogenation and the overall substrate scope of these enzymes has delayed substrate expansion and engineering efforts.This thesis approaches this gap in knowledge through the leveraging of a variety of interdisciplinary techniques including biochemistry, bioinformatics, analytical mass spectrometry, organic synthesis, microbiology, and structural biology. Chapter 2 will discuss our efforts to bioinformatically define the sequence space occupied by selective VHPOs, the substrates we can expect them to react with, the sequence determinants of selectivity, and a chemical method to detect selective VHPO activity in the absence of an organic acceptor substrate. Chapter 3 will discuss our method development to increase the accessibility the naphthoquinone-meroterpenoid VHPO, NapH4, and our discovery and characterization of a novel meroterpenoid chlorinating VHPO, LvcH. Chapter 4 will discuss the identification and characterization of a unique sub-family of selective VHPOs that brominate alkyl quinolone quorum sensor molecules. We utilize phylogenetics, in vitro biochemistry, and MALDI-MSI imaging of co-cultures to identify that this sub-family catalyzes bromination of exogenous substrates as a general detoxification mechanism. Chapter 5 will discuss our investigation into the mechanism of substrate selectivity in selective VHPOs. Through cryo-EM structural resolution, mutagenesis, and biophysical methods we identify that selective VHPOs achieve substrate selectivity through the separation of vanadate-dependent halide oxidation and substrate halogenation into separate pockets.Through the work performed in this thesis, we have provided a significant advancement to the understanding of selective VHPOs that will be applied to future biocatalytic optimization of selective VHPOs and to the genome mining of novel natural product therapeutics. Through our studies of the basic chemistry of selective VHPOs we have developed chemical probes to detect haloamine reactive intermediates, advanced specific techniques to apply them to studying the VHPOs, and discovered an incredible untapped potential of enzymatic chemistry that will provide an intellectual starting point for projects in the lab moving forward.