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A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their

Sandeepchowdary Bala1, Bharati Reddi1, Anthony Addlagatta1

  • 1Division of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, Telangana, India; Academy of Scientific and Innovative Research (AcSIR), Kamla Nehru Nagar, Ghaziabad, Uttar Pradesh 201002, India.

Biochimica Et Biophysica Acta. Proteins and Proteomics
|November 17, 2022
PubMed

Insights

Methionine aminopeptidase (MetAP) from Pyrococcus furiosus (PfuMetAP2a) cleaves leucine, unlike other known MetAPs. This archaeal enzyme is also resistant to ovalicin inhibition due to active site differences.

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Methionine aminopeptidases (MetAPs) are crucial enzymes that remove the N-terminal methionine from newly synthesized proteins.
  • MetAPs are classified into Type 1 and Type 2, with eukaryotes possessing both types, while prokaryotes and archaea have only one type.
  • Previously, it was believed that MetAPs only cleave methionine, but crude extracts of archaeal PfuMetAP2a showed potential activity against leucine.

Purpose of the Study:

  • To investigate the enzymatic activity of purified recombinant Pyrococcus furiosus Type 2a methionine aminopeptidase (PfuMetAP2a).
  • To determine if PfuMetAP2a exhibits activity against leucine substrates.
  • To understand the differential inhibition of PfuMetAP2a by ovalicin, a known Type 2 MetAP inhibitor.

Main Methods:

  • Purification of recombinant PfuMetAP2a.
  • Enzymatic assays to test cleavage of methionine and leucine substrates.
  • Inhibition assays using the natural product ovalicin.
  • Bioinformatic analysis of active site residues and structural comparisons.

Main Results:

  • Purified PfuMetAP2a directly cleaves leucine in addition to methionine.
  • PfuMetAP2a is significantly less sensitive to inhibition by ovalicin compared to eukaryotic Type 2 MetAPs.
  • Bioinformatic analysis revealed that residue N53 in PfuMetAP2a, analogous to G222 in human MetAP2b, alters the active site orientation via interaction with H62, reducing ovalicin affinity.
  • A mutation N53G in PfuMetAP2a partially restored ovalicin sensitivity.

Conclusions:

  • PfuMetAP2a possesses a broader substrate specificity than previously thought, cleaving both methionine and leucine.
  • Structural differences in the active site, particularly the role of residue N53 and its interaction with H62, explain the resistance of PfuMetAP2a to ovalicin.
  • These findings highlight evolutionary divergence in MetAP active sites and their inhibitor interactions.
Keywords:
ArchaeaInhibitionMethionine aminopeptidaseOvalicinPyrococcus furiosusSubstrate specificityType 2 MetAP

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