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

Abstract

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 (Ap 3 A), 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 Mg 2؉ or Mn 2؉ . For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap 3 A, the T4 enzyme hydrolyzes higher homologues including Ap 4 -6 A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. The Nudix hydrolase superfamily consists of widely distributed subfamilies of enzymes characterized by a shared structural motif: the Nudix box having a highly conserved consensus sequence,where "X " represents any amino acid, and U usually represents one of the bulky, hydrophobic amino acids, Ile, Leu, or Val (1). Enzymological and structural studies have shown that this arrangement of amino acids leads to a novel loop-helix-loop motif involved in substrate binding and catalysis (2-8). A recent BLAST (9) search of the data banks for the Nudix signature sequence has revealed over 800 open reading frames from more than 200 species including archaea, eubacteria, and eukarya, and at present, ϳ50 of the gene products have been identified. These are all enzymes active on nucleoside diphosphate derivatives including (deoxy)nucleoside triphosphates, coenzymes, cell signaling molecules, and nucleotide sugars (1), and we have been systematically identifying and characterizing new members of the superfamily.An examination of the T4 bacteriophage genome reveals one gene, e

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis

“…Here, we showed that chloroplastic AtNUDX23 had FAD pyrophosphohydrolase activity, by which FAD was hydrolyzed to FMN and AMP (Tables I and II). Nudix hydrolases having FAD pyrophosphohydrolase activity have been identified in bacteria and bacteriophage (Xu et al, 2002;Tirrell et al, 2006). Recently, the characteristics of a bifunctional enzyme (AtFMN/FHy) with activities of riboflavin kinase and FAD synthetase in Arabidopsis were reported (Sandval and Roje, 2005).…”

“…Reaction product(s) FMN and/or AMP were detected (data not shown). The K m value for FAD (9.1 6 0.9 mM) was markedly lower than that of Paenibacillus thiaminolyticus YZGD (Tirrel et al, 2006) and T4 bacteriophage nudE.1 (Xu et al, 2002;Table II). On the other hand, the enzyme showed low affinity (386 6 76 mM) for ADP-Rib.…”

Bacteriophage T4 Genome

“…T4 biology will "bootstrap" us to the recognition of similar biochemical processes in related phage while identifying genes and proteins that are novel to VOL. 67, 2003 BACTERIOPHAGE T4 GENOME 131 (1204), and ORF 24.1 (rnlB) encodes a second T4 RNA ligase related to the RNA-editing enzymes of trypanosomes and those present in sequenced Archaea (426). T4 will be a handy genetic and biochemical tool for detailed studies of their respective enzymatic activities.…”

The NudA Protein in the Gastric Pathogen Helicobacter pylori Is an Ubiquitous and Constitutively Expressed Dinucleoside Polyphosphate Hydrolase

“…The nucleoside diphosphate linked to some other moiety X (Nudix) protein family, formerly called the MutT protein family, consists of about 800 proteins in more than 200 species from all kingdoms (1). This group of proteins may be divided into subfamilies based on the substrate specificity: ADP-ribose, dinucleoside polyphosphates, NADH, nucleotide sugars, or ribo-and deoxyribonucleoside triphosphates (2).…”