Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata

Abstract

Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K = 2960 ± 940 μM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCP, suggesting that the other site, TCP, remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCP binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.

Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature

“…S10. Multiple previous structures of DHP-B have been determined under cryogenic conditions (de Serrano et al, 2010;McCombs, Moreno-Chicano et al, 20.17;Carey et al, 2018). Despite the B factors being smaller for structures determined at 100 K compared with those reported at RT here, the overall structural features are maintained.…”

Rational design of heme enzymes for biodegradation of pollutants toward a green future

“…Recently, Ghiladi and coworkers [62] showed that DHP B exhibited a catalytic efficiency (k cat /K m ) of 2,100 M −1 Sec −1 in oxidation of 2,4,6-TCP, which is 10-fold higher than that of WT DHP A ( Table 2). Replacement of Phe21 in the heme distal site of DHP B by Trp was found to decrease the catalytic efficiency by 7.2-fold, and more pronounced effect (decreased by 15fold) was observed for the F21W mutation in DHP A (Table 2) [63].…”

Peroxidase versus Peroxygenase Activity: Substrate Substituent Effects as Modulators of Enzyme Function in the Multifunctional Catalytic Globin Dehaloperoxidase

“…Non-His-tagged DHP B was overexpressed and purified as described in the literature. ,, DHP B crystals were obtained using the hanging-drop vapor diffusion method. The crystals were grown from mother liquor solutions of 32% PEG 3350 and 0.2 M ammonium sulfate at pH 6.4 and equilibrated against identical reservoir solutions.…”

“…These peroxide-activated forms of DHP initially return to the ferric resting state upon substrate oxidation, although further reduction to oxyferrous DHP has been noted under some conditions. 24,25,34,40,41 Given the diverse functions (O 2 carrier, peroxidase, peroxygenase, and oxidase) exhibited by DHP across this broad panel of substrates, we have previously suggested that the substrate itself can act to modulate enzyme activity in DHP, 29,30 with binding/orientation (relative to the ferryl iron), pK a , and redox potential all being key determinants in terms of which mechanism leads to substrate oxidation. More interestingly, however, it also appears that substituent effects may play a pseudoallosteric role (Figure 1): the peroxidase substrate phenol is converted to a peroxygenase substrate upon addition of a 4-nitro substituent, 34 whereas 4-Br-phenol is an inhibitor 42 of peroxidase and peroxygenase activities in DHP.…”

“…Inhabiting coastal mudflats that are rich in biogenically produced halometabolites excreted from other organisms, A. ornata faces stressors that include brominated indoles, pyrroles, phenols, andof particular relevance to this studyguaiacols. To overcome the challenges posed by this diverse array of toxic haloaromatic compounds, A. ornata employs its hemoglobin to function both as a detoxification enzyme and as an O 2 -transport protein. − Named dehaloperoxidase (DHP), this coelomic hemoglobin possesses an unusually broad substrate profile encompassing halophenols, , haloindoles, and (halo)­pyrroles and as such is the first known multifunctional catalytic globin to possess biologically relevant peroxidase − and peroxygenase ,, activities, as well as an oxidase activity that leads to the formation of indigo derivatives. DHP also exhibits activity against compounds of anthropogenic origin, including the H 2 O 2 -dependent oxidation of mono- and dinitrophenols, and is also capable of binding azole species, including imidazole, benzotriazole, benzimidazole, and indazole.…”