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.

The Origins and Roles of Methylthiolated Cytokinins: Evidence From Among Life Kingdoms

“…The presence of CK metabolites, or the genetic mechanisms required for CK production, have been reported in all kingdoms of life (Björk, 1995;Mayaka et al, 2019). Cytokinin biosynthesis involves conserved mechanisms even among evolutionary distant organisms, and the formation of CKs in bacteria, fungi, plants, or mammals all involve specific gene homologs and their corresponding enzymes, including IPT, adenosine kinase (AK), LONELY GUY (LOG) and a CK-degradation enzyme, cytokinin oxidase/dehydrogenase (CKX; Sakakibara, 2006;Chanclud et al, 2016;Trdá et al, 2017;Daudu et al, 2019).…”

Cytokinin Sensing in Bacteria

“…In conclusion, we are currently witnessing tremendous advances in the field of CK signaling in microorganisms. These recent discoveries must also be put in perspective of the explosion in the number of identified LONELY GUY genes in many different phyla, attesting to the extent of the action spectrum of CKs within the tree of life [24,25]. These data provide a foundation for investigations that will substantially increase our knowledge of the broad roles of these ancient molecules in regulating intra-and interspecific communications.…”

The Hulks and the Deadpools of the Cytokinin Universe: A Dual Strategy for Cytokinin Production, Translocation, and Signal Transduction

“…Notably, the activity with AMP was higher than with di- and triphosphates of CKs. Bacterial LOGs [ 138 , 139 , 140 , 141 ], as well as Archeal LOG [ 142 ], were shown to hydrolyse AMP. Their activity towards CK nucleotides was not estimated in vitro, with the exception of the Mycobacterial enzyme.…”