Bioinformatics-based hypotheses

To investigate the hypotheses about the structural mechanisms of selectivity in VHPOs, as well as validate ASR as a technique for generating VHPOs, we decided to use an ASR tool, FireprotASR (Musil et al). This program is capable of generating a phylogenetic tree based on a user-inputted query sequence by pulling homologous proteins from multiple databanks, along with other customization options that streamline use.

6 sequences (or “nodes”) from the ancestral reconstruction were chosen as representatives of the respective clades, 3 sequences that represent distinct, non-selective ancestors, and 3 that represent selective ancestors.

Ancestral node 177 (N177) was generated as the common ancestor for VHPOs that tend to modify meroterpenoid scaffold molecules (red), a class of molecules highly relevant for natural product research. N160 was generated as the ancestor for alkyl quinolone modifying VHPOs (blue), molecules which are used in bacterial signaling pathways. N153 serves as a general ancestor for all selective VHPOs. The nonselective ancestors N220, N241, and N291 were chosen due to the distinct clades they define.

To further investigate the chosen ancestral VHPOs, alphaFold and CAVER analysis were used to make initial models. One unique characteristic of selective VHPOs is the existence of a secondary internal pocket structure, mediated by a conserved lysine residue. The primary pocket is the site of vanadate binding to a histidine residue, and also the site of halide oxidation. Previous work in our lab has shown that this secondary pocket is responsible for the selectivity for organic substrates. The models are shown above, with selective VHPOs in box A and nonselectives in box B. They are compared to crystal structures of characterized VHPOs.

One interesting result from this analysis was that the ancestral enzymes from the selective side of the phylogenetic tree displayed this secondary pocket, whereas the nonselectives had a lack of a distinct pocket. In addition, the conserved lysine residue in selective VHPOs is replaced with a proline in the nonselectives. This suggests that the selective enzymes will be able to make a haloamine bond and perform selective halogenation, while the nonselectives will oxidize the halide to form hypohalous acid and immediately release it.