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Defining Substrate Specificities for Lipase and Phospholipase Candidates
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Structural basis of substrate specificity and selectivity of murine cytosolic 5'-nucleotidase III

Christina L Grobosky1, Jennifer B Lopez, Sarahbeth Rennie

  • 1Department of Chemistry, Georgian Court University, Lakewood, NJ 08701, USA.

Journal of Molecular Biology
|August 29, 2012
PubMed

Insights

Cytosolic 5'-nucleotidase III (cN-III) deficiency causes hemolytic anemia by preventing pyrimidine nucleotide breakdown. This study reveals the enzyme's structure bound to uridine 5'-monophosphate, explaining its specificity.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Cytosolic 5 -nucleotidase III (cN-III) selectively degrades pyrimidine 5 -monoribonucleotides during red blood cell maturation.
  • Deficiency in cN-III activity leads to nonspherocytic hemolytic anemia due to pyrimidine nucleotide accumulation in erythrocytes.
  • The precise mechanisms of cN-III substrate specificity and its role in erythrocyte function remain incompletely understood.

Purpose of the Study:

  • To determine the first X-ray structure of a catalytically inactivated murine cN-III variant bound to its natural substrate, uridine 5 -monophosphate.
  • To elucidate the atomic details of the enzyme-substrate complex and understand the molecular basis for cN-III's specificity.
  • To investigate the role of specific amino acid residues and interactions in substrate recognition and binding.

Main Methods:

  • X-ray crystallography of a catalytically inactivated murine cN-III variant.
  • Co-crystallization with uridine 5 -monophosphate.
  • Structure determination at 1.74Å resolution.
  • Structure and sequence analysis coupled with enzymatic characterization of mutant variants.

Main Results:

  • The X-ray structure reveals a closed conformation of cN-III upon binding uridine 5 -monophosphate.
  • Specific interactions, including π-stacking with Trp113 and His68, T-shaped stacking with Tyr114, and polar contacts with Thr66 and Ser117, stabilize the nucleotide's nitrogenous base.
  • Hydrogen bonds involving two water molecules and residues Asp72 and His68, along with interactions with Glu96 for ribose recognition, are crucial for nucleotide binding.
  • The structure provides atomic-level insights into cN-III's specificity for pyrimidine 5 -nucleotides over purine 5 -nucleotides.

Conclusions:

  • The determined structure provides a detailed molecular explanation for cN-III's selective degradation of pyrimidine 5 -monoribonucleotides.
  • Understanding these interactions is key to comprehending the pathogenesis of cN-III deficiency-related hemolytic anemia.
  • This structural information can guide future research on enzyme function and the development of therapeutic strategies.

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