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Lonely Guy(LOG)proteins are important enzymes in cellular organisms.They catalyze the final step in the production of biological active cytokinin via dephosphoribosylation reaction.LOGs are vital enzymes in plants for activation of cytokinins,which are crucial for plant growth and development.In fungi LOGs play roles in fungi-plant interactions with diverse effects on the host plants ranging from non-pathogenic to pathogenic relationships.Also similar phenomena are encountered in plant-bacteria interactions,thus the LOGs are enzymes which produces cytokinins that enhance virulence capability and leading to formation of disease symptoms in plants by some pathogenic bacteria species.On the other hand,it was established that accumulation of cytokinin degraded products is lethal to proteasomal-deficient Mycobacterium tuberculosis,a human obligate pathogen.SiRe_0427was annotated as a homologue of a DNA processing protein A(DprA)which is involved in DNA transformation and recombination.We initially tested if SiRe_0427has the properties as a DprA:DNA binding and interaction with DNA recombinase RadA.We did not detect neither of DNA binding nor interaction with RadA activities.Instead,this study has identified the SiRe_0427as a LOG homologue in Sulfolobus islandicus REY15A,a hyperthermophilic archaeon via the biochemical analysis.The protein possesses the LOG"GGGxGTxxE"-motif,located between(α4-β5)within which the enzyme active site residues are found.This study employed the same technique used in the previous studies on LOGs from eukaryotes and bacteria,using thin layer chromatography(TLC),detected a phosphoribohydrolase activity of wild type(WT)on AMP as the substrate.Also found that the mutant proteins of either catalytic or adenosine5-monophosphate(AMP)binding sites lost their phosphoribohydolase activity,similar to other known LOGs.Further,it is also determined that the oligomeric status of SiRe_0427as a homotetramer in solution using size exclusion chromatography and a chemical cross-linking experiments,which is in agreement to its modeling structure.Therefore,the finding of this study provides the experimental evidences for the presence of LOG homologue in archaea and in addition it nullifies the notion that SiRe_0427is DprA protein.It should be noted with importance that the LOGs need to be reclassified.Based on the previous classification,this work proposed that an additional cluster should be added giving total of three clusters i.e.,clusterⅠ(dimers),clusterⅡ(tetramers)and clusterⅢ(hexamers).It is obvious that SisLOG exhibited high heat resistance properties and a tetramer status indicates that SisLOG is a new lineage(branch)of the LOG protein domains.
We also performed genetic analysis of SisLOG.We found that the gene encoding SiRe_0427can be knocked out.Although sire_0427mutant exhibits slight difference in growth pattern in liquid and solid media compared to the wild type.It does not show any difference in sensitivity to UV irradiation under our laboratory growth conditions,in agreement with the result of the biochemical analysis.We assumed that archaeal LOGs may function in generation of secondary messengers from either cytokinin-like precursor,similar as in classical LOGs or nucleotide analogs,such as circular NMPs.Further study is needed to reveal the substrate and in vivo function of archaeal LOGs.
To our knowledge this is the first report showing archaeal LOG homologue is active as a phosphoribohydrolase.Understanding the cellular role(s)of LOG proteins and its possible cytokinin varieties will provide basis for its applications in the fields such as agriculture,industry and/or as drug target in pathogenic microbes.
We also performed genetic analysis of SisLOG.We found that the gene encoding SiRe_0427can be knocked out.Although sire_0427mutant exhibits slight difference in growth pattern in liquid and solid media compared to the wild type.It does not show any difference in sensitivity to UV irradiation under our laboratory growth conditions,in agreement with the result of the biochemical analysis.We assumed that archaeal LOGs may function in generation of secondary messengers from either cytokinin-like precursor,similar as in classical LOGs or nucleotide analogs,such as circular NMPs.Further study is needed to reveal the substrate and in vivo function of archaeal LOGs.
To our knowledge this is the first report showing archaeal LOG homologue is active as a phosphoribohydrolase.Understanding the cellular role(s)of LOG proteins and its possible cytokinin varieties will provide basis for its applications in the fields such as agriculture,industry and/or as drug target in pathogenic microbes.