No abstract

Nucleoside 5-O-phosphorothioates are formed in vivo as primary products of hydrolysis of oligo(nucleoside phosphorothioate)s (PS-oligos) that are applied as antisense therapeutic molecules. The biodistribution of PS-oligos and their pharmacokinetics have been widely reported, but little is known about their subsequent decay inside the organism. We suggest that the enzyme responsible for nucleoside 5-O-monophosphorothioate ((d)NMPS) metabolism could be histidine triad nucleotide-binding protein 1 (Hint-1), a phosphoramidase belonging to the histidine triad (HIT) superfamily that is present in all forms of life. An additional, but usually ignored, activity of Hint-1 is its ability to catalyze the conversion of adenosine 5-O-monophosphorothioate (AMPS) to 5-O-monophosphate (AMP). By mutagenetic and biochemical studies, we defined the active site of Hint-1 and the kinetic parameters of the desulfuration reaction (P-S bond cleavage). Additionally, crystallographic analysis (resolution from 1.08 to 1.37 Å ) of three engineered cysteine mutants showed the high similarity of their structures, which were not very different from the structure of WT Hint-1. Moreover, we found that not only AMPS but also other ribonucleoside and 2-deoxyribonucleoside phosphorothioates are desulfurated by Hint-1 at the following relative rates: GMPS > AMPS > dGMPS > CMPS > UMPS > dAMPS Ͼ Ͼ dCMPS > TMPS, and during the reaction, hydrogen sulfide, which is thought to be the third gaseous mediator, was released. (Fig. 1). 5Ј-O-Phosphorothioates of ribonucleosides (NMPSNMPS and dNMPS (together denoted (d)NMPS)) are formed during the enzymatic hydrolysis of oligo(nucleoside phosphorothioate) (PS-oligos) that contain a sulfur atom attached in non-bridging positions to the phosphorus atom at each or selected internucleotide bond(s). Synthetic PS-oligos have been developed as antisense probes for genomic research and medicinal applications (1, 2). These oligonucleotides are promising therapeutic molecules because they are much more stable against nucleolytic degradation in blood and various cellular systems than their natural, unmodified counterparts (3-5). Their hydrolysis in plasma, kidney, and liver proceeds mainly from the 3Ј end, resulting in the appearance of the mononucleoside 5Ј-phosphorothioates identified in urine from PS-oligo-injected animals (6, 7). (d)NMPS may exert cytotoxic effects affecting cell proliferation, DNA or RNA synthesis, and other unknown processes (8, 9). Recently, the phosphorothioate DNA segments have been identified in bacterial DNA (10), which makes investigations into PS-oligo metabolism even more important.Although several reports have been published on the biodistribution of PS-oligos, little is known about the metabolism of the products of their degradation in vivo. It has been suggested that extracellular dNMPS and dNMP can be converted to the corresponding nucleoside by 5Ј-nucleotidase (ecto-5Ј-NT) (9). This membrane-bound enzyme preferably releases adenosine from extracellular AMP, but other purine and pyrimid...

PDB Reference: hHINT1-AMP complex, 3tw2.Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1-adenosine 5 0 -monophosphate (AMP) complex at 1.38 Å resolution obtained from a new monoclinic crystal form is reported. The final structure has R cryst = 0.1207 (R free = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine-triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the first complete structure of the rabbit HINT1-adenosine complex is reported at 1.10 Å resolution, which is one of the highest resolutions obtained for a HINT1 structure. The final structure has an R(cryst) of 14.25% (R(free) = 16.77%) and the model exhibits good stereochemical qualities. A detailed analysis of the atomic resolution data allowed an update of the details of the protein structure in comparison to previously published data.