Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family

Abstract

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. In this work, we expand on our recent phylogenetic inference of the entire RNR family and describe the evolutionarily relatedness of insertions and extensions around the structurally homologous catalytic barrel. Using evo‐velocity and sequence similarity network (SSN) analyses, we show that the N‐terminal regulatory motif known as the ATP‐cone domain was likely inherited from an ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, we also show that the C‐terminal extensions of class II RNRs can contain folded domains that share homology with an Fe‐S cluster assembly protein. Finally, using sequence analysis and AlphaFold2, we show that the sequence motif of a catalytically essential insertion known as the finger loop is tightly coupled to the catalytic mechanism. Based on these results, we propose an evolutionary model for the diversification of the RNR family.

Activity modulation in anaerobic ribonucleotide reductases: nucleotide binding to the ATP-cone allosterically mediates substrate binding to the active site

“…The amino acid sequence of PcNrdD suggests that it belongs to the formate-requiring class III RNRs (Burnim et al , 2022b; Wei et al ., 2014), and initial optimization of the assay composition showed that this was the case (Figure 1 - figure supplement 1). ATP-cone mediated activation of PcNrdD enzyme activity by ATP or inhibition by dATP was tested with two different substrates.…”

Conformational Landscapes of a Class I Ribonucleotide Reductase Complex during Turnover Reveal Intrinsic Dynamics and Asymmetry

“…Ribonucleotide reductases (RNRs) are essential enzymes in all free-living organisms, as they enable the only de novo synthesis pathway of 2′-deoxyribonucleotides, the building blocks for DNA ( 1 ). Despite wide evolutionary diversity ( 2 , 3 ), all RNRs share a common catalytic mechanism initiated by the formation of a thiyl radical on a conserved active-site cysteine ( 4 ). The oxygen-dependent class I RNRs ( 5 , 6 ) are used by aerobic life, including most eukaryotes, many prokaryotes, and associated viruses ( 2 , 3 ).…”

“…Despite wide evolutionary diversity ( 2 , 3 ), all RNRs share a common catalytic mechanism initiated by the formation of a thiyl radical on a conserved active-site cysteine ( 4 ). The oxygen-dependent class I RNRs ( 5 , 6 ) are used by aerobic life, including most eukaryotes, many prokaryotes, and associated viruses ( 2 , 3 ). These RNRs have long fascinated researchers for their relevance as drug targets and for their dynamic quaternary structure involving two subunits (Figure 1A): 1) a small ferritin-like subunit (β) that houses a stable radical cofactor, and 2) a large catalytic subunit (ɑ) that contains the nucleotide-binding sites, including the active site, an allosteric site that controls substrate specificity, and additional allosteric sites involved in regulating overall enzyme activity.…”

Leveraging ancestral sequence reconstruction for protein representation learning

“…Another limitation of traditional ASR methods for generating functional sequences is that insertions and deletions (indels) and mishandled or ignored by maximum likelihood sequence evolution models. As indel events are often responsible for driving functional diversification [46][47][48][49] , we also developed an automated pipeline for the maximum likelihood reconstruction of insertions/deletions (indels) in large ancestral sequence databases produced with mASR (SI Fig 1) . For site n, all tips are assigned a binary label (0 for no indel, 1 for indel), depending on whether a gap character is observed.…”