Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The E. coli derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs has been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the lower structural robustness of EcTyrRS relative to MjTyrRS. We show that engineered TyrRS mutants in general exhibit significantly lower thermostability relative to their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homolog from a thermophilic bacteria, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

The Lonely Guy (LOG) Homologue SiRe_0427 from the Thermophilic Archaeon Sulfolobus islandicus REY15A Is a Phosphoribohydrolase Representing a Novel Group

ABSTRACT Lonely Guy (LOG) proteins are important enzymes in cellular organisms, which catalyze the final step in the production of biologically active cytokinins via dephosphoribosylation. LOG proteins are vital enzymes in plants for the activation of cytokinin precursors, which is crucial for plant growth and development. In fungi and bacteria, LOGs are implicated in pathogenic or nonpathogenic interactions with their plant hosts. However, LOGs have also been identified in the human pathogen Mycobacterium tuberculosis, and the accumulation of cytokinin-degraded products, aldehydes, within bacterial cells is lethal to the bacterium in the presence of nitric oxide, suggesting diverse roles of LOGs in various species. In this study, we conducted biochemical and genetic analysis of a LOG homologue, SiRe_0427, from the hyperthermophilic archaeon Sulfolobus islandicus REY15A. The protein possessed the LOG motif GGGxGTxxE and exhibited phosphoribohydrolase activity on adenosine-5-monophosphate (AMP), similar to LOGs from eukaryotes and bacteria. Alanine mutants at either catalytic residues or substrate binding sites lost their activity, resembling other known LOGs. SiRe_0427 is probably a homotetramer, as revealed by size exclusion chromatography and chemical cross-linking. We found that the gene encoding SiRe_0427 could be knocked out; however, the Δsire_0427 strain exhibited no apparent difference in growth compared to the wild type, nor did it show any difference in sensitivity to UV irradiation under our laboratory growth conditions. Overall, these findings indicate that archaeal LOG homologue is active as a phosphoribohydrolase. IMPORTANCE Lonely Guy (LOG) is an essential enzyme for the final biosynthesis of cytokinins, which regulate almost every aspect of growth and development in plants. LOG protein was originally discovered 12 years ago in a strain of Oryza sativa with a distinct floral phenotype of a single stamen. Recently, the presence of LOG homologues has been reported in Mycobacterium tuberculosis, an obligate human pathogen. To date, active LOG proteins have been reported in plants, pathogenic and nonpathogenic fungi, and bacteria, but there have been no experimental reports of LOG protein from archaea. In the current work, we report the identification of a LOG homologue active on AMP from Sulfolobus islandicus REY15A, a thermophilic archaeon. The protein likely forms a tetramer in solution and represents a novel evolutionary lineage. The results presented here expand our knowledge regarding proteins with phosphoribohydrolase activities and open an avenue for studying signal transduction networks of archaea and potential applications of LOG enzymes in agriculture and industry.

Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei

Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1–Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3′ end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR–Cas systems.

The DNA-binding protein HTa fromThermoplasma acidophilumis an archaeal histone analog

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are phylogenetically widespread, often highly abundant, but not universal: several archaeal lineages have lost histone genes from their coding repertoire. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we use micrococcal nuclease digestion followed by high-throughput sequencing (MNase-Seq) to elucidate primary chromatin architecture in an archaeon without histones, the acido-thermophilic archaeonThermoplasma acidophilum. We confirm and extend prior results showing thatT. acidophilumharbours a HU family protein, HTa, that is highly expressed and protects a sizeable fraction of the genome from MNase digestion. Charting HTa-based chromatin architecture across the growth cycle and comparing it to that of three histone-encoding archaea (Methanothermus fervidus, Thermococcus kodakarensisandHaloferax volcanii), we then present evidence that HTa is an archaeal histone analog. HTa-protected fragments are GC-rich, display histone-like mono- and dinucleotide patterns around a conspicuous dyad, exhibit relatively invariant positioning throughout the growth cycle, and show archaeal histone-like oligomerization dynamics. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

CandidatusNitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first cultivated thermophilic representative with a sequenced genome, which allows to search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota.CandidatusNitrosocaldus cavascurensis has been cultivated from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 25h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome ofCa.N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase, genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the importance of this compound for AOA metabolism.Ca.N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position

ABSTRACT The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii. Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.