Adenosine 5′-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties

Abstract

Adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25+/-1 kDa. It catalyses the hydrolysis of a nucleoside 5'-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5'-tetraphosphate (p4A) is 8.2, Vmax is 21+/-1.7 micromol/min per mg of protein and the Km for p4A is 3+/-0.6 microM. At saturating p4A concentrations, the rate constant for the reaction is 8.5+/-0.7 s-1 [at 30 degrees C, in 50 mM Hepes/KOH (pH8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5'-tetraphosphate are hydrolysed at the same rate. Adenosine 5'-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively.

NudC Nudix hydrolase from Pseudomonas syringae, but not its counterpart from Pseudomonas aeruginosa, is a novel regulator of intracellular redox balance required for growth, motility and biofilm formation

“…DC3000 (designated here Ps NudC) and NudC from P. aeruginosa PAO1161 (designated Pa NudC) belong to the widely distributed Nudix pyrophosphatase family, characterized by the presence of a highly conserved Nudix motif GX 5EX7REUXEEXGU (where U is usually Ile, Leu or Val). Nudix enzymes hydrolyse, albeit with different degrees of specificity, a variety of nucleoside diphosphate derivatives including: canonical and modified forms of dNTPs, NTPs (Kamiya et al, 2001;Nunoshiba et al, 2004;Ito et al, 2005), nucleotide sugars (Moreno-Bruna et al, 2001;Gabelli et al, 2002;, diadenosine polyphosphates (Guranowski et al, 1997;Ismail et al, 2003;Olejnik et al, 2007), several coenzymes (Xu et al, 2000;Gasmi and McLennan, 2001;Kang et al, 2003), m 7 GTP mRNA cap (Dunckley and Parker, 1999) and 5′-triphosphorylated bacterial RNA transcripts (Deana et al, 2008) The majority of Nudix substrates are either potentially toxic or constitute metabolic intermediates whose concentrations require modulation during cell cycle. Thus, it has been postulated that Nudix hydrolases function in cells as 'housecleaning' enzymes (Bessman et al, 1996).…”

Diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus L.) belongs to a novel group of specific metallophosphatases

“…The plant cell also produces specific phosphatases showing distinct metabolic functions, e.g. phosphoenolpyruvate phosphatase [9], phosphotyrosyl‐protein phosphatase [10], adenosine 5′‐tetraphosphate phosphohydrolase [11], pyrophosphatase [12,13], or phytase [14]. A widely distributed pyrophosphatase/phosphodiesterase activity has recently been found in plants that catalyze the hydrolytic breakdown of several NDP‐monosaccharides [15,16].…”

4-Coumarate:Coenzyme A Ligase Has the Catalytic Capacity to Synthesize and Reuse Various (Di)Adenosine Polyphosphates

“…However, the existence of highly specific enzymes that catalyze the hydrolysis of (di)nucleoside polyphosphates, such as (asymmetrical) dinucleoside tetraphosphatase (EC 3.6.1.17), dinucleoside triphosphatase (EC 3.6.1.29), and nucleoside 5Ј-tetraphosphatase (EC 3.6.1.14), indicates that these nucleotide derivatives may exist and have a function in planta (Jakubowski and Guranowski, 1983;Guranowski et al, 1996Guranowski et al, , 1997. In this report, we demonstrate that Arabidopsis 4CL2, a key enzyme of plant secondary metabolism, can catalyze the synthesis of various mono-and dinucleoside polyphosphates.…”