Making sense of a missense mutation: characterization of MutT2, a Nudix hydrolase from Mycobacterium tuberculosis, and the G58R mutant encoded in W-Beijing strains of M. tuberculosis.
Recent polymorphism analyses of Mycobacterium tuberculosis strains have identified missense mutations unique to the W-Beijing lineage in genes belonging to the Nudix hydrolase superfamily. This study investigates the structure and function of one of these Nudix hydrolases, MutT2, and examines the effect that the W-Beijing mutation (G58R) has on enzyme characteristics. MutT2 has a preference for cytidine triphosphates, and although the G58R mutation does not alter nucleotide specificity, it reduces the protein's affinity for divalent cations. The K(D) of free Mg(2+) is 79-fold higher for the G58R mutant (3.30 +/- 0.19 mM) compared with that for the wild-type (41.7 +/- 1.4 microM). Circular dichroism and nuclear magnetic resonance spectroscopy measurements show that while the mutation does not perturb the overall structure of the protein, protein stability is significantly compromised by the presence of the arginine with DeltaG (H(2)O), the free-energy of unfolding, being reduced by 2.48 kcal mol(-1) in the G58R mutant. Homology modeling of MutT2 shows that Gly-58 is in close proximity (10.8 A) to the Mg(2+) binding site formed by the highly conserved Nudix box residues and hydrogen bonds with Ala-54 in the preceding alpha-helix. This may explain the increased divalent cation requirement and decreased stability observed when an arginine is substituted for glycine at this position. A role for MutT2 in the regulation of cytidine-triphosphates available for nucleotide-dependent reactions is postulated, and the impact that the G58R mutation may have on these reactions is discussed.
Moreland
Nicole J
NJ
Maurice Wilkins Centre for Molecular Biodiscovery and Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, New Zealand. n.moreland@mac.com
Charlier
Caroline
C
Dingley
Andrew J
AJ
Baker
Edward N
EN
Lott
J Shaun
JS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006868:Hydrolysis; D008274:Magnesium / Q000502:physiology; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D017433:Protein Structure, Secondary / Q000235:genetics; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000096964:Nudix Hydrolases
D001120:Arginine; D002413:Cations, Divalent; D003570:Cytidine Triphosphate; D005998:Glycine; D008274:Magnesium; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases