Abstract
Three members of the Nudix (nucleoside diphosphate X) hydrolase superfamily have been cloned from Escherichia coli MG1655 and expressed. The proteins have been purified and identified as enzymes active on nucleoside diphosphate derivatives with the following specificities. Orf141 (yfaO) is a nucleoside triphosphatase preferring pyrimidine deoxynucleoside triphosphates. Orf153 (ymfB) is a nonspecific nucleoside tri- and diphosphatase and atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. Orf191 (yffH) is a highly active GDP-mannose pyrophosphatase. All three enzymes require a divalent cation for activity and are optimally active at alkaline pH, characteristic of the Nudix hydrolase superfamily. The question of whether or not Orf1.9 (wcaH) is a bona fide member of the Nudix hydrolase superfamily is discussed.
Highlights
SCHEME 1 where X represents any amino acid, and U is usually a bulky hydrophobic amino acid, such as Leu, Val, or Ile
We have discovered a superfamily of enzymes related by the similarity of their substrates and defined by a highly conserved sequence of amino acids, the “Nudix box”3 as follows
This amino acid motif is of highly predictive value, because proteins containing the Nudix box invariably catalyze the hydrolysis of nucleoside diphosphates linked to some other moiety, X, the acronym, Nudix [1], in a few cases other organic compounds containing pyrophosphate linkages are hydrolyzed
Summary
A major tool in annotating or relating a protein to a function is the recognition of structural motifs or amino acid signature sequences In this vein, we have discovered a superfamily of enzymes related by the similarity of their substrates and defined by a highly conserved sequence of amino acids, the “Nudix box” as follows,. This amino acid motif is of highly predictive value, because proteins (enzymes) containing the Nudix box invariably catalyze the hydrolysis of nucleoside diphosphates linked to some other moiety, X, the acronym, Nudix [1], in a few cases other organic compounds containing pyrophosphate linkages are hydrolyzed At this writing, over 2000 members of the superfamily from 200 species ranging from viruses to humans have been identified through BLAST [2] searches, and we have been systematically cloning, expressing, and characterizing their activities. Amino acids in the Nudix box could be designated as follows: G1N, X5N, E7N, R15N, etc., and an amino acid substitution by alanine, for example, would be designated as G1NA, X5NA, E7NA etc. according to standard convention
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