Enzymatic characteristics of Nudix hydrolase 2 (Nud2), an 8-oxo-dGTP hydrolase from <i>Myxococcus xanthus</i>

Abstract

Myxococcus xanthusNudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn 2+ was the most effective cofactor for stimulating oxidized deoxynucleotide hydrolase activity. The K m of Nud2 with 8-oxo-dGTP for Mn 2+ was 19-fold lower than that for Mg 2+ , and was 2-fold lower than that with dGTP for Mn 2+ . The specificity constant (k cat /K m ) for 8oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif ( 84 AX 5 90 GX 7 REX 2 EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.Nudix hydrolases are a family of pyrophosphatases containing the highly conserved 23-residue sequence segment (GX 5 EX 7 REUXEEXGU, where U is a bulky hydrophobic amino acid such as Ile, Leu, or Val, and X is any amino acid) called the Nudix motif . They are metal-dependent enzymes found in all classes of organism, and can hydrolyze a wide range of organic pyrophosphates, including nucleoside di-and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars, RNA caps, NAD(P)H, coenzyme A, and guanosine-3¢,5¢-tetraphosphate (ppGpp) with varying degrees of substrate specificity (McLennan, 2006). As these substrates are involved in energy metabo-8-oxo-dGTP hydrolase of M. xanthus

Characterization of glutamate‐cysteine ligase and glutathione synthetase from the δ‐proteobacterium Myxococcus xanthus

“…50 However, Mn 2+ is required for the growth and survival of bacteria, and the intracellular Mn 2+ levels are homeostatically regulated. 51 Several of M. xanthus enzymes that we have studied are activated by Mn 2+ but not by Mg 2+ (e.g., protein phosphatases, acetyl-CoA synthase, aminoacyl-tRNA synthetases, and Nudix hydrolases), [52][53][54][55][56][57][58] suggesting that the concentration of intracellular Mn 2+ in M. xanthus should be sufficient to support catalytic activity.…”