SummaryAll 21 of the Nudix hydrolase genes from the radiation-resistant organism Deinococcus radiodurans have been cloned into vectors under the control of T7 promoters and expressed as soluble proteins in Escherichia coli. Their sizes range from 9.8 kDa (91 amino acids) to 59 kDa (548 amino acids). Two novel proteins were identified, each with two Nudix boxes in its primary structure, unique among all other known Nudix hydrolases. Extracts of each of the expressed proteins were assayed by a generalized procedure that measures the hydrolysis of nucleoside diphosphate derivatives, and several enzymatic activities were tentatively identified. In addition to representatives of known Nudix hydrolase subfamilies active on ADP-ribose, NADH, dinucleoside polyphosphates or (deoxy)nucleoside triphosphates, two new enzymes, a UDP-glucose pyrophosphatase and a CoA pyrophosphatase, were identified.
The genome of Bacillus cereus contains 26 Nudix hydrolase genes, second only to its closest relative, Bacillus anthracis which has 30. All 26 genes have been cloned, 25 have been expressed, and 21 produced soluble proteins suitable for analysis. Substrates for 16 of the enzymes were identified; these included ADP-ribose, diadenosine polyphosphates, sugar nucleotides, and deoxynucleoside triphosphates. One of the enzymes was a CDP-choline pyrophosphatase, the first Nudix hydrolase active on this substrate. Furthermore, as a result of this and previous work we have identified a new subfamily of the Nudix hydrolase superfamily recognizable by a specific amino acid motif outside of the Nudix box.We have been studying a superfamily of enzymes catalyzing the hydrolysis of nucleoside diphosphates linked to some other moiety, x. These "Nudix" hydrolases (1) are widely distributed in nature with representatives in eukaryotes, prokaryotes, archaea, viruses, and at least one mycoplasm. The identifying feature of the superfamily is a pattern of nine highly conserved amino acids called the Nudix box, embedded in a sequence of 23 amino acids, GX 5 EX 7 REUXEEXGU SCHEME 1 in which U is a hydrophobic amino acid, usually Ile, Leu, or Val, and X may be any amino acid. To date, more than 1800 open reading frames from over 360 species have been uncovered by BLAST searches (2) of the sequenced genomes, data banks, and expressed sequence tags. On the basis of their specificities, the individual members of the superfamily may be assigned to subfamilies active on various nucleoside triphosphates, nucleotide sugars, coenzymes such as NADH, FAD, or CoA, cell signaling molecules, e.g. dinucleoside polyphosphates, and toxic metabolites such as ADP-ribose and mutagenic and toxic nucleotides. The number of Nudix hydrolase genes varies widely among species, and there is presently neither a predictor nor algorithm foretelling how many of these genes might be expected in a given organism. In Table I, the number of Nudix genes, ascertained by examining open reading frames, is compared by genome size. Deinococcus radiodurans, the most radiation-resistant organism known, attracted our attention, because at that time it had the largest number of Nudix hydrolase genes of any organism, almost twice that of Escherichia coli, although D. radiodurans has a smaller genome. We cloned all 21 genes of D. radiodurans and expressed and partially characterized several interesting new enzymes of the Nudix hydrolase superfamily (3). Since then, the genome of Bacillus anthracis has been sequenced (4), and it encodes 30 Nudix hydrolase genes, the largest number found in any organism to date. Characterization of these gene products would almost certainly uncover new members of the superfamily, and perhaps contribute to our understanding of the physiology of this interesting pathogenic microbe. However, our research with this organism is proscribed for several reasons. Fortunately, the genome of Bacillus cereus, a very closely related organism (4, 5), has just been ...
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