Structure of human cytosolic X-prolyl aminopeptidase: a double Mn(II)-dependent dimeric enzyme with a novel three-domain subunit

J Biol Chem. 2008 Aug 15;283(33):22858-66. doi: 10.1074/jbc.M710274200. Epub 2008 May 30.

Abstract

X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6 angstroms. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopeptidases / chemistry*
  • Aminopeptidases / genetics
  • Aminopeptidases / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Cytosol / enzymology*
  • Genetic Variation
  • Humans
  • Kinetics
  • Manganese / metabolism*
  • Models, Molecular
  • Protein Conformation
  • Protein Subunits / chemistry

Substances

  • Protein Subunits
  • Manganese
  • Aminopeptidases
  • X-Pro aminopeptidase