Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis . Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understanding its molecular basis in such inhospitable conditions, given that such conditions lead to loss of structural changes and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments. We present the state of recent progress in extreme environmental genomics. We then present an overview of our current understanding of the biomolecular adaptation to extreme conditions. As our current and future understanding of biomolecular structure–function relationships in extremophiles requires methodologies adapted to extremes of pressure, temperature, and chemical composition, advances in instrumentation for probing biophysical properties under extreme conditions are presented. Finally, we briefly discuss possible future directions in extreme biophysics. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6,779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade Ø. We employed small-angle X-ray scattering (SAXS), cryogenic-electron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from Synechococcus phage S-CBP4 and report the most minimal RNR architecture to-date. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study.

Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH 3 -H 4 folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S -adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli  MetH have shown that this flexible, multidomain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a “divide and conquer” approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH 3 -H 4 folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.