Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases

Abstract

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5′-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating β subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y 356 [β], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y 356 • along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any β metallocofactor highlights the adaptability of the 10-stranded αβ barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.