WenLian Xu

The Nudix hydrolases of Deinococcus radiodurans

All 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 Rickettsia prowazekii Invasion Gene Homolog (invA) Encodes a Nudix Hydrolase Active on Adenosine (5′)-pentaphospho-(5′)-adenosine

The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (NpnN; n ≥ 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5′)-pentaphospho-(5′)-adenosine into ATP and ADP with a Km of 0.1 mm and kcat of 1.9 s−1. Guanosine (5′)-pentaphospho-(5′)-guanosine and adenosine-(5′)-hexaphospho (5′)-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg2+ or Zn2+, for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.

The gene e.1 (nudE.1) of T4 bacteriophage designates a new member of the Nudix hydrolase superfamily active on flavin adenine dinucleotide, adenosine 5'-triphospho-5'-adenosine, and ADP-ribose.

The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5′-triphospho-5′-adenosine (Ap3A), and ADP-ribose. Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg2+or Mn2+. For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap3A, the T4 enzyme hydrolyzes higher homologues including Ap4–6A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. Although the protein was predicted to be orthologous to E. coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following. (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates. (b) The e.1 gene does not complement mutT1 in vivo. (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage. The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of thenudE gene, and we therefore propose the mnemonicnudE.1 for the T4 phage orthologue.

Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: A member of the Nudix family

Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg(2+), its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.

Three new Nudix hydrolases from Escherichia coli.

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.

A New Subfamily of the Nudix Hydrolase Superfamily Active on 5-Methyl-UTP (Ribo-TTP) and UTP

A new subfamily of the Nudix hydrolases, identified by conserved amino acids upstream and downstream of the Nudix box, has been characterized. The cloned, expressed, and purified orthologous enzymes have major activities on the non-canonical nucleoside triphosphate 5-methyl-UTP (ribo-TTP) and the canonical nucleotide UTP. In addition to their homologous signature sequences and their similar substrate specificities, the members of the subfamily are inhabitants of or are related to the bacterial rhizosphere. We propose the acronym and mnemonic, utp, for the gene designating this unique UTPase.

Legionella pneumophila NudA Is a Nudix Hydrolase and Virulence Factor

ABSTRACT We studied the identity and function of the 528-bp gene immediately upstream of Legionella pneumophila F2310 ptsP (enzyme INtr). This gene, nudA, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as Escherichia coli YgdP, in that ApnA’s, in particular diadenosine pentaphosphate (Ap5A), were the preferred substrates. NudA hydrolyzed Ap5A to ATP plus ADP. Both ptsP and nudA were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene nudA was present in 19 of 20 different L. pneumophila strains tested and in 5 of 10 different Legionella spp. other than L. pneumophila. An in-frame nudA mutation was made in L. pneumophila F2310 to determine the phenotype. The nudA mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25°C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with nudA. The nudA mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with L. pneumophila pneumonia showed that the nudA mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in L. pneumophila and is a virulence factor.

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

Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli. The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates. Whereas all other nucleoside triphosphatase members of the superfamily release inorganic pyrophosphate and the cognate nucleoside monophosphate, YtkD hydrolyses nucleoside triphosphates in a stepwise fashion through the diphosphate to the monophosphate, releasing two molecules of inorganic orthophosphate. Contrary to a previous report, our enzymological and genetic studies indicate that ytkD is not an orthologue of E. coli mutT.

Structural studies of the Nudix GDP-mannose hydrolase from E. coli reveals a new motif for mannose recognition

The Nudix hydrolase superfamily, characterized by the presence of the signature sequence GX5EX7REUXEEXGU (where U is I, L, or V), is a well‐studied family in which relations have been established between primary sequence and substrate specificity for many members. For example, enzymes that hydrolyze the diphosphate linkage of ADP‐ribose are characterized by having a proline 15 amino acids C‐terminal of the Nudix signature sequence. GDPMK is a Nudix enzyme that conserves this characteristic proline but uses GDP‐mannose as the preferred substrate. By investigating the structure of the GDPMK alone, bound to magnesium, and bound to substrate, the structural basis for this divergent substrate specificity and a new rule was identified by which ADP‐ribose pyrophosphatases can be distinguished from purine‐DP‐mannose pyrophosphatases from primary sequence alone. Kinetic and mutagenesis studies showed that GDPMK hydrolysis does not rely on a single glutamate as the catalytic base. Instead, catalysis is dependent on residues that coordinate the magnesium ions and residues that position the substrate properly for catalysis. GDPMK was thought to play a role in biofilm formation because of its upregulation in response to RcsC signaling; however, GDPMK knockout strains show no defect in their capacity of forming biofilms. Proteins 2011;

The pnhA Gene of Pasteurella multocida Encodes a Dinucleoside Oligophosphate Pyrophosphatase Member of the Nudix Hydrolase Superfamily

The pnhA gene of Pasteurella multocida encodes PnhA, which is a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. PnhA hydrolyzes diadenosine tetra-, penta-, and hexaphosphates with a preference for diadenosine pentaphosphate, from which it forms ATP and ADP. PnhA requires a divalent metal cation, Mg(2+) or Mn(2+), and prefers an alkaline pH of 8 for optimal activity. A P. multocida strain that lacked a functional pnhA gene, ACP13, was constructed to further characterize the function of PnhA. The cellular size of ACP13 was found to be 60% less than that of wild-type P. multocida, but the growth rate of ACP13 and its sensitivity to heat shock conditions were similar to those of the wild type, and the wild-type cell size was restored in the presence of a functional pnhA gene. Wild-type and ACP13 strains were tested for virulence by using the chicken embryo lethality model, and ACP13 was found to be up to 1,000-fold less virulent than the wild-type strain. This is the first study to use an animal model in assessing the virulence of a bacterial strain that lacked a dinucleoside oligophosphate pyrophosphatase and suggests that the pyrophosphatase PnhA, catalyzing the hydrolysis of diadenosine pentaphosphates, may also play a role in facilitating P. multocida pathogenicity in the host.