Characterization of enzymes

Monochlorodimedone assay

Initial halide specificity assays were carried out using a spectrophotometer to measure changes in absorbance. The basis of this assay is that monochlorodimedone (MCD) reacts with diffusible hypochlorous (HOCl) and hypobromous acid (HOBr) to halogenate a specific position on the molecule, as shown in Wever et. al. This halogenation disrupts isomerization, and eliminates the molecules’ absorbance of 290 nm wavelength light, giving a read-out for the enzymes’ release of hypohalous acids. Each enzyme stock was diluted to 1 mg/mL concentration for this assay.

The assay is split into 3 regimes: (i) Baseline absorbance in the presence of KCl and vanadate before the reaction can proceed, (ii) Addition of hydrogen peroxide to start reaction and measure release of HOCl, and (iii) Addition of KBr to measure HOBr release. Absorbance of 290 nm light is measured every 10 seconds over the course of 30 minutes. Each color on the plot corresponds to a different replicate run.

Structure and reaction of MCD with a hypohalous acid releasing enzyme

N177, N220, and N160 were all shown to be capable HOBr producers, while N153 showed some HOBr production, and N291 showed very minor HOBr release. N160’s results are contrary to our clade-based hypothesis of reactivity since its descendents are selective bromoperoxidases, which sequester HOBr upon bromide oxidation (rather than freely diffusing it into solution). N241 was not tested due to poor yield in the purification step.

While the MCD assay is useful for showing which halides each enzyme is capable of oxidizing and releasing, it is incapable of detecting which halides are oxidized and then coordinated within the enzyme.

Substrate assays

To assess substrate halogenation capacity, we performed an organic substrate panel assay on N153, N160, N177, and N220. N291 and N241 were omitted due to low yields. Based on previous experiments, extant members of the clade under N177 were capable of chlorinating small amounts of alkyl quinolone molecules, so they are used as a readout for chlorination capability. MHQ, HHQ, and HQNO were chosen due to their differences in functional groups. In addition, Lavanducyanin (LVC) was also added as a meroterpenoid readout.

Organic substrate panel

The conversion of substrates to their chlorinated forms was measured using LCMS and peak integration of starting material and products after normalization to assess percent conversion. Two time points were taken, at 2 and 22 hours.

Both N153 and N177 showed selective patterns of chlorination, whereas N160 and N220 did not display any chlorination of the substrates. We were expecting N160 to show chlorination as it was releasing HOBr in the MCD assay, but it’s possible this construct is lacking certain structural features that enable HOCl sequestration. N153 shows a general ability to chlorinate substrates, with HQNO and LVC being the primary targets. N177 shows a greater preference for LVC chlorination overall, and a reduced ability to chlorinate the alkyl quinolones. These distributions line up with the bioinformatic predictions of N153 being a general, selective VHPO and N177 having evolved to act more on meroterpenoid substrates.

A bromination panel was conducted, and all substrates were quickly converted to their brominated forms within 2 hours. This is likely due to diffused HOBr, as the enzymes were shown to release it in the MCD assay. N220 did not display any chlorination, supporting its classification as a nonselective bromoperoxidase. Based on these results, we can conclude that N153 and N177 are selective chloroperoxidases.

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