No abstract

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3′→5′ mRNA decay. Some previous studies suggested that both m7GpppG and m7GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m7GDP (7-methylguanosine diphosphate) and m32,2,7GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5′→3′ mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode and yeast). Furthermore, whereas mononucleoside diphosphates (m7GDP and m32,2,7GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m7GTP and m32,2,7GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m7GTP and m32,2,7GTP are cleaved at a slower rate than their corresponding dinucleotides (m7GpppG and m32,2,7GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m7GDP, they have a high binding affinity for m7GDP and m7GDP potently inhibits DcpS hydrolysis of m7GpppG, suggesting that m7GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m7GDP in mRNA metabolic pathways, due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

The activity of the Caenorhabditis elegans scavenger decapping enzyme (DcpS) on its natural substrates and dinucleotide cap analogs, modified with regard to the nucleoside base or ribose moiety, has been examined. All tested dinucleotides were specifically cleaved between β‐ and γ‐phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis (Km, Vmax) were determined using fluorescence and HPLC methods, as complementary approaches for the kinetic studies of C. elegans DcpS. From the kinetic data, we determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We showed that m32,2,7GpppG and m32,2,7GpppA are cleaved with higher rates than their monomethylated counterparts. However, the higher specificity of C. elegans DcpS for monomethylguanosine caps is illustrated by the lower Km values. Modifications of the first transcribed nucleotide did not affect the activity, regardless of the type of purine base. Our findings suggest C. elegans DcpS flexibility in the first transcribed nucleoside‐binding pocket. Moreover, although C. elegans DcpS accommodates bulkier groups in the N7 position (ethyl or benzyl) of the cap, both 2′‐O‐ and 3′‐O‐methylations of 7‐methylguanosine result in a reduction in hydrolysis by two orders of magnitude.

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.

Edited by Hans Eklund Keywords:Histidine triad-family protein Dual catalytic activity Adenosine 5 0 -phosphosulfate hydrolysis Adenosine 5 0 -phosphosulfate phosphorolysis a b s t r a c t Histidine triad (HIT)-family proteins interact with different mono-and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5 0 -phosphosulfate (APS or SO 4 -pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.