31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

Abstract

Eukaryotic cells encode AMP-lysine (AMP-N-⑀-(N-␣acetyl lysine methyl ester) 5-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotidebinding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative 31 P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5-monophosphoramidate (AMP-NH 2 ). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 M KCl, 0.9 M NaCl, 0.5 M NaOAc, or 10 mM MnCl 2 . Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

“…Construction of Plasmids-The DHFR fusion protein was constructed based on the pJLCF15DmH plasmid template previously constructed in our laboratory (2,26). The pJLCF15DmH plasmid encodes a fusion protein containing a FLAG peptide at the N terminus of L54F-E. coli DHFR, linked by a 15-amino acid linker to hHint1.…”

“…In the first step, the active site is nucleotidylated by nucleophilic attack at the substrate phosphorous by a conserved active site histidine (His 112 for hHint1 (6). X-ray crystal structure and size exclusion chromatography analyses have revealed that hHint1 exists as a homodimer (2,23,24). Similar to other homodimeric enzymes, such as bacterial alkaline phosphatase, each monomer contains a well separated active site that does not participate in the dimer interface (25).…”

“…Hints are homodimeric proteins that have been found to be purine phosphoramidases (2)(3)(4)(5). Recently, we have demonstrated that lysyl-adenylate (lysyl-AMP) generated by bacterial and human lysyl-tRNA synthetases (LysRS) are also substrates for the respective bacterial and human Hints (6).…”

“…Recently, we have demonstrated that lysyl-adenylate (lysyl-AMP) generated by bacterial and human lysyl-tRNA synthetases (LysRS) are also substrates for the respective bacterial and human Hints (6). Escherichia coli hinT knock-out mutants exhibited salt-dependent cell growth, whereas Hint1 knock-out mice have an increased susceptibility to 7, 12-dimethylbenz[a]anthracene (DMBA)-induced ovarian and mammary tumors by the carcinogen DMBA and increased incidence of spontaneous tumors (2,7,8). In addition, Hint1 has been shown to have a potential role on cancer cells apoptosis and p53 expression (9).…”

“…: 612-625-2614; Fax: 612-624-0139; E-mail: wagne003@umn.edu. 2 The abbreviations used are: Hint, histidine triad nucleotide-binding protein; hHint1, human histidine triad nucleotide-binding protein 1; AIPA, adenosine 5Ј-indole-3-propionic adenylate; AMPPCP, adenosine 5Ј-(␣,␤-methylenediphosphate); HIT, histidine triad; MTX, methotrexate; r.m.s. deviation, root mean square deviation; SEC, size exclusion chromatography; TpAd, tryptamine adenosine phosphoramidate monoester; Ap 3 A, diadenosine P 1 ,P 3 -triphosphate; Fhit, fragile histidine triad; GalT, galactose-1-phosphate uridyltransferase; DHFR, dihydrofolate reductase.…”