The 26 Nudix Hydrolases of Bacillus cereus, a Close Relative of Bacillus anthracis

Abstract

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 ...

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

“…B. cereus CDP-Chase was cloned into a pET-24a vector as described previously (45). Plasmid DNA was used to transform E. coli BL21(DE3) cells.…”

“…These changes affect a salt bridge present in the structures of other Nudix enzymes between residues E 7 N and R 15 N of the Nudix signature sequence (Fig. 1) (45). To gain insight into the structural and functional consequences of these differences, as well as into the relationship between sequence and specificity, we investigated the kinetics and determined the crystal structure of the B. cereus CDP-Chase.…”

“…The anthrax pathogen Bacillus anthracis contains the highest number of Nudix genes of any sequenced organism (45). To take advantage of this extensive repertoire, the Nudix enzymes from the closely related but less pathogenic Bacillus cereus were cloned and expressed as a way of discovering enzymes that act on novel Nudix substrates.…”

Gene ytkD of Bacillus subtilis Encodes an Atypical Nucleoside Triphosphatase Member of the Nudix Hydrolase Superfamily

“…In fact, the E. coli and human enzymes have a higher V max on dGTP than on 8-oxo-dGTP. Second, there is very little amino acid identity between YtkD and E. coli MutT, whereas there is a high degree of identity between YtkD and a protein that was recently characterized from Bacillus cereus (20). This is illustrated in Fig.…”

Modulation of the Poly(ADP-ribosyl)ation Reaction via the Arabidopsis ADP-Ribose/NADH Pyrophosphohydrolase, AtNUDX7, Is Involved in the Response to Oxidative Stress

“…Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases catalyze the hydrolysis of intact and oxidatively damaged nucleoside diphosphates and triphosphates, nucleotide sugars, coenzymes, dinucleoside polyphosphates, and RNA caps in various organisms such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al, 1996;Xu et al, 2004;Kraszewska, 2008). We have previously reported the characteristics of cytosolic Nudix hydrolases (AtNUDX1-AtNUDX11) in Arabidopsis (Arabidopsis thaliana; Ogawa et al, 2005).…”