Selenocysteine Substitution in a Class I Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) employ a complex radical-based mechanism during nucleotide reduction involving multiple active site cysteines that both activate the substrate and reduce it. Using an engineered allo-tRNA, we substituted two active site cysteines with distinct function in the class Ia RNR of Escherichia coli for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the C 439 U α 2 mutant protein interactions with nucleotide substrates and the cognate β 2 subunit demonstrates that the endogenous Y 122• of β 2 is reduced under turnover conditions, presumably through radical transfer >35 Å to form a transient U 439• species. This putative U 439• species is formed in a kinetically competent fashion, but is incapable of initiating nucleotide reduction via 3′-H abstraction. An analogous C 225 U α 2 protein is also capable of radical transfer from Y 122• , but the radical-based substrate chemistry partitions between turnover and stalled reduction akin to the reactivity of mechanism-based inhibitors of RNR. The results collectively demonstrate the essential role of cysteine redox chemistry in the class I RNRs and establish a new tool for investigating thiyl radical reactivity in biology.

Introducing Selenocysteine into Recombinant Proteins in Escherichia coli

“…Together, these four genes ( As SelA, allo‐tRNA UTu2D , As SelD, and Td Trx1; cloned together in the pSecUAG‐Evol2 plasmid) provide the most efficient EF‐Tu‐dependent Sec insertion system described to date (Mukai et al., 2018). It has been used for the expression of a variety of hydrogenases and dehydrogenases (unpublished data from our group) and a class I ribonucleotide reductase (Greene, Stubbe, & Nocera, 2019).…”

Radicals in Biology: Your Life Is in Their Hands

“…Their importance has firmly been established within RNRs by trapping them with bond formation to adjacent residues, allowing for their detection when subsequent transformations are blocked . In the absence of such protein protective mechanisms, advanced methods will be needed to uncover them in biology (Figure A,D,E), including, among others, site-specific incorporation of seleno­cysteine using UAA engineering . Development of these methods will provide ways to further probe the importance of amino acid C • and other radical-mediated transformations, many of which will involve unprecedented chemistries, as with RNRs.…”

Site‐Specific Selenocysteine Incorporation into Proteins by Genetic Engineering

“…mechanistic studies of its active site cysteines. [11] A recent work reported the use of allo-tRNA for the replacement of Sec to each of the Ni-coordinating cysteine residues in the oxygentolerant E. coli [NiFe]-hydrogenase-1 (Hyd-1) for functional studies. [37] 3.5.…”

“…For example, the catalytic activity of Sec-containing MsrB1 is ~800-fold higher than the Cys-variants; [6] substitution of Sec in the active site of mammalian TrxR by Cys leads to 100-fold lower k cat and 10-fold lower K M in reduction of thioredoxin; [7] and substitution of Sec to Cys in mammalian Gpx4 renders neuron cells susceptible to ferroptotic cell death due to the hydroperoxide-induced inactivation of the Gpx4-Cys variant. [8] The unique properties of Sec also provide opportunities for the engineered selenoproteins to be applied in various areas, such as designing artificial enzymes, [9] enhancement of protein redox sensitivity, [10] for mechanistic studies of enzyme active sites, [11] being NMR probes to study ligand-protein binding, [12] being a redox-active tag for affinity purification, [13] promotion of oxidative protein folding, [14] and facilitation of expressed protein ligation. [15] However, recombinant producing selenoproteins with high Sec specificity is difficult, making the precise functions of many selenoproteins remain poorly characterized, thus efficient approaches for heterologous production of selenoproteins are in high demand.…”