Cloning, expression and characterization of YSA1H, a human adenosine 5'-diphosphosugar pyrophosphatase possessing a MutT motif.
The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K(m) and k(cat) values with ADP-ribose were 60 microM and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg(2+) or 100-250 microM Mn(2+) ions; fluoride was inhibitory, with an IC(50) of 20 microM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.
Gasmi
L
L
School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB, U.K.
Cartwright
J L
JL
McLennan
A G
AG
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000247:Adenosine Diphosphate Sugars / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D000096964:Nudix Hydrolases
D000245:Adenosine Diphosphate Glucose; D000246:Adenosine Diphosphate Ribose; D000247:Adenosine Diphosphate Sugars; D015226:Dinucleoside Phosphates; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases; C012276:diadenosine pyrophosphate; C403715:NUDT5 protein, human; C019620:ADPribose pyrophosphatase