Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins

Abstract

Proteins that have a histidine triad in their active sites belong to the HIT-protein superfamily. They are ubiquitous, are involved in the metabolism of different nucleotides and catalyze their hydrolysis and/or phosphorolysis liberating either the corresponding 5 0 -NMP or 5 0 -NDP, respectively. We studied substrate specificity of nine recombinant HIT-proteins with adenosine 5 0 -phosphosulfate (1), adenosine 5 0 -phosphoramidate (2), adenosine 5 0 -phosphorothioate (3), adenosine 5 0 -phosphorofluoride (4), diadenosine 5 0 ,5 0 0 0 -P 1 ,P 3 -triphosphate (5), di(7-methylguanosine) 5 0 ,5 0 0 0 -P 1 ,P 3 -triphosphate (6) and adenosine 5 0 -hypophosphate (7). Preferences for the recognition of these compounds as substrates by individual proteins differed. All the proteins hydrolyzed (1) but the Arabidopsis thaliana Hint1 did it very slowly. None of the proteins cleaved (7). Only A. thaliana Hint1 and Escherichia coli HinT hydrolyzed (3). Three proteins known as dinucleoside triphosphatases, human and A. thaliana Fhit-proteins and Trypanosoma brucei HIT-45, cleaved (1), ( 2), ( 4), ( 5) and ( 6). Caenorhabditis elegans decapping protein DcpS degraded (1), ( 5), ( 6) and poorly (4). A. thaliana aprataxin-like protein and Hint4 hydrolyzed only (1), ( 2) and ( 4), in that order of efficiency. Velocities of those reactions and some K m values were determined. Applicability of this study to the metabolism of certain nucleotidyl-derivatives is discussed.

Histidine Triad Nucleotide-binding Protein 1 (HINT-1) Phosphoramidase Transforms Nucleoside 5′-O-Phosphorothioates to Nucleoside 5′-O-Phosphates

“…Recently, Hint enzymes from other organisms (Hint from E. coli and Hint-1 from Arabidopsis thaliana) have been shown to be able to desulfurate the AMPS (46). Conclusion-The discovery of the first phosphorothioate backbone modification of DNA in vivo (10) and the use of synthetic PS-oligos in medicinal applications give rise to questions about the function and metabolism of this modification.…”

Exogenous adenosine 5′-phosphoramidate behaves as a signal molecule in plants; it augments metabolism of phenylpropanoids and salicylic acid in Arabidopsis thaliana seedlings

“…We trust that our findings will be followed by different "omic" experiments to shed more light on the physiological functions of NH 2 -pA and/or its synthetic analog F-pA. Bearing in mind that NH 2 -pA and F-pA are very good substrates for various HIT-proteins that act as nucleoside phosphoramidases (Guranowski et al, 2008(Guranowski et al, , 2010b, we suggest that these ubiquitous proteins may assist in signal transduction from these compounds to factors that control expression of the genes highlighted above. This process might be accompanied by adenylylation of a target molecule and the energy needed for that could come from the splitting of the NeP or FeP bonds.…”

“…Recently, we have found the same activity in extracts of mammalian tissues (unpublished) and from yellow lupin seeds (Wojdyła-Mamo n and Guranowski, 2014). The supposition that NH 2 -pA is a ubiquitous compound and that its concentration is enzymatically controlled may be supported by the existence of various proteins that catalyze the cleavage of NH 2 -pA to ammonia and either 5 0 -AMP (pA), by hydrolysis (Kuba et al, 1994;Bieganowski et al, 2002;Guranowski et al, 2010aGuranowski et al, , 2010bGuranowski et al, , 2011Bretes et al, 2013), or 5 0 -ADP (ppA) by phosphorolysis (Guranowski et al, 2010a). Both the synthesis (Wojdyła-Mamo n and Guranowski, 2014) and degradation (Guranowski et al, 2010b) of NH 2 -pA can be controlled by HIT proteins.…”

“…Pursuing our studies of the enzymes involved in the metabolism of unusual nucleotides, we have focused on the biochemistry of adenosine 5 0 -monophosphoramidate (NH 2 -pA) (Guranowski et al, 2008(Guranowski et al, , 2010a(Guranowski et al, , 2010b. This compound is believed to occur in all organisms.…”

Phosphorothioate Nucleotides and Oligonucleotides – Recent Progress in Synthesis and Application

“…Stec and co-workers have reported that a human enzyme Fhit (also from the HIT family) is able to conduct the same process [181]. Guranowski et al studied activity of nine recombinant HIT proteins towards AMPS [211]. The proteins represented four branches of the HIT superfamily: three Fhit proteins (human, Arabidopsis thaliana, and Trypanosoma brucei), four Hint proteins (Hint1, Hint3, and Hint4 from A. thaliana, and HinT from Escherichia coli), one decapping protein from Caenorhabditis elegans, and aprataxin-like protein from A. thaliana.…”