Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases

Abstract

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

Figure 1. Sequence alignment of TmPep1050 and CelM vs. the characterized M42 aminopeptidases.

Characterized M42 aminopeptidases used for the multiple alignment: PhTET1, PhTET2, and PhTET3 from P. horikoshii , , , HmTET from Haloarcula marismortui , SpPepA from S. pneumoniae , SthPep1079 and SthPep1080 from Symbiobacterium thermophilum , and YsdC (pdb code 1VHE) from Bacillus subtilis . * and • indicate, respectively, conserved amino acids and residues with similar properties. ♦ indicates amino acids involved in metal ion binding. Amino acids constituting the S1 pocket defined from structural studies of SpPepA and PhTET2 are highlighted in black boxes.

Figure 2. Influence of pH on the LAP activity of (A) TmPep1050 and (B) CelM.

Curves of activity vs. pH in the MES (dashed line), MOPS (solid line), and HEPES (dash-dotted line) buffers. Specific activities (sp act) are expressed in µmol of p-nitroaniline produced by hydrolysis of the amino acid-pNA derivative per minute and per µmol of enzyme.

Figure 3. Influence of temperature on the LAP activity of TmPep1050 (closed circles) and CelM (open circles).

(A) Activity-vs.-temperature plot; (B) Plot showing the logarithm of the activity vs. the inverse of the temperature. Specific activities (sp act) are expressed in µmol of p-nitroaniline produced by hydrolysis of the amino acid-pNA derivative per minute and per µmol of enzyme. Trend lines were calculated by linear regression (R² = 0.9735 for TmPep1050, and R² = 0.9853 for CelM).