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. 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 the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue.
Highlights
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, GXXXXXEXXXXXXXREUXEEXGU
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
The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of the nudE gene, and we propose the mnemonic nudE.1 for the T4 phage orthologue
Summary
The expression plasmid pET-24a(ϩ) (Kmr) and E. coli HMS174 (DE3) were from Novagen (Madison, WI). Phages L1/a (a lysozyme amber mutant, referred to as eam in the text) and rII NB 3034 were from the collection of Larry Gold, University of Colorado, Boulder. T4 strains used as templates for cloning the e.1 gene were gifts of Linda Reha-Krantz, University of Alberta, Canada, and Lindsay W. The plasmid pTrc99A (Ampr) and Sephadex G-100 were from Amersham Biosciences, and the restriction enzymes, PCR kits, calf intestinal alkaline phosphatase, and inorganic pyrophosphatase were from Stratagene (La Jolla, CA). The abbreviations used are: Ap3A or diadenosine triphosphate, adenosine 5Ј-triphospho-5Ј-adenosine; ApnA, diadenosine polyphosphates; Kmr, kanamycin resistance; Ampr, ampicillin resistance. The gel filtration sizing column, HiLoadTM 16/60 SuperdexTM 75 was from Amersham Biosciences
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