Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction

Abstract

Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced Thermus spp. genome. Phylogenetic analysis revealed a distant origin of the nrdJm gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GC-content, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B12-dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60–70 °C, which is in the range of the growth optimum of Thermus spp. Experiments in combination with the Thermus thermophilus thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence.

A Novel One-Pot Enzyme Cascade for the Biosynthesis of Cladribine Triphosphate

“…The published optimal reaction temperatures of PPK and RNR are 70 °C for both enzymes [29,32]. For APT, the optimal reaction temperature was determined to be 50 °C in a preliminary experiment ( Figure A1A).…”

“…Enzyme engineering of the PPK would be a possible way to reduce this side reaction as well. Formation of 2Cl-dADP by RNR catalyzed 2CL-ADP reduction can be excluded as a source for dADP generation, since the applied enzyme is a strict nucleoside triphosphate reducing RNR [32].…”

“…Examples are uracil phosphoribosyltransferases (UPT) or hypoxanthine(-guanine) phosphoribosyltransferases (H(G)PT) [17,38,41,42]. PPKs and RNRs are capable of the conversion of different natural nucleotides, with both purine and pyrimidine bases [29,32,35]. Therefore, a certain degree of substrate promiscuity towards non-natural nucleotides can be expected from both enzymes.…”

“…They catalyze the reduction of nucleotides to the corresponding deoxyribonucleotides and are responsible for the de novo biosynthesis of the DNA building blocks in nature [31]. Depending on the class of RNR, they catalyze the reduction of either nucleoside di-or triphosphates [32][33][34]. In principle, they are capable of reducing different natural nucleotides but the substrate specificity is subject to allosteric regulation.…”

Genomic and Seasonal Variations among Aquatic Phages Infecting the Baltic Sea GammaproteobacteriumRheinheimerasp. Strain BAL341

“…For example, the RNR genes within the barba phages shared low sequence similarity to the host’s RNR genes (23% for nrdA and 22% for nrdB ) (Table 2). In general, phages have the same RNRs as their host, but the opposite has also been noted in several instances (60, 82) and has been suggested to be a sign of either horizontal gene transfer within phages with extended host range or of a shifted host range in phages over longer time periods (83), which potentially could be the case for these genes in the barba phages.…”