Journal
JOURNAL OF INORGANIC BIOCHEMISTRY
Volume 217, Issue -, Pages -Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2021.111370
Keywords
Peroxidases; de novo proteins; Heme; Redox enzymology; Biomolecular simulation
Funding
- BBSRC [BBI014063/1, BB/R016445/1, BB/M025624/1, BB/M009122/1 SWDTP]
- SynBioCDT (EPSRC and BBSRC Centre for Doctoral training in Synthetic Biology) [EP/L016494/1]
- EPSRC [EP/M022609/1]
- BBSRC [BB/R016445/1, BB/L01386X/1] Funding Source: UKRI
- EPSRC [EP/J010588/1, EP/G007705/1, EP/L016494/1, EP/M022609/1] Funding Source: UKRI
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The de novo enzyme C45 exhibits extensive catalytic promiscuity, with substrate activity primarily determined by the redox potential of the substrate and the active oxidising species in peroxidase chemistry. Substrate:protein interactions play a significant role in determining electron transfer rates from substrate to heme, affecting the kinetic parameters of the enzyme. Biomolecular simulation identified potential interactions and binding sites near the heme cofactor, providing insight into the catalytic workings of C45.
The design and construction of de novo enzymes offer potentially facile routes to exploiting powerful chemistries in robust, expressible and customisable protein frameworks, while providing insight into natural enzyme function. To this end, we have recently demonstrated extensive catalytic promiscuity in a heme-containing de novo protein, C45. The diverse transformations that C45 catalyses include substrate oxidation, dehalogenation and carbon?carbon bond formation. Here we explore the substrate promiscuity of C45?s peroxidase activity, screening the de novo enzyme against a panel of peroxidase and dehaloperoxidase substrates. Consistent with the function of natural peroxidases, C45 exhibits a broad spectrum of substrate activities with selectivity dictated primarily by the redox potential of the substrate, and by extension, the active oxidising species in peroxidase chemistry, compounds I and II. Though the comparison of these redox potentials provides a threshold for determining activity for a given substrate, substrate:protein interactions are also likely to play a significant role in determining electron transfer rates from substrate to heme, affecting the kinetic parameters of the enzyme. We also used biomolecular simulation to screen substrates against a computational model of C45 to identify potential interactions and binding sites. Several sites of interest in close proximity to the heme cofactor were discovered, providing insight into the catalytic workings of C45.
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