Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

peer reviewedThe crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate

The Cold-Active M1 Aminopeptidase from the Arctic Bacterium Colwellia psychrerythraea

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Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.

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.info:eu-repo/semantics/publishe

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

<p>Curves of activity vs. pH in the MES <b>(</b>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.</p

Characterization of the endoglucanase activity of TmPep1050 and CelM.

<p>Activities are expressed in µmol reduced sugar produced by substrate hydrolysis and were estimated by the DNS method. SEM is given for each value.</p

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

<p>(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).</p

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

<p>Characterized M42 aminopeptidases used for the multiple alignment: PhTET1, PhTET2, and PhTET3 from <i>P. horikoshii </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Dur1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Dur2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Russo1" target="_blank">[34]</a>, HmTET from <i>Haloarcula marismortui </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Franzetti1" target="_blank">[19]</a>, SpPepA from <i>S. pneumoniae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Kim1" target="_blank">[20]</a>, SthPep1079 and SthPep1080 from <i>Symbiobacterium thermophilum </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Kumaki1" target="_blank">[25]</a>, and YsdC (pdb code 1VHE) from <i>Bacillus subtilis </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050639#pone.0050639-Kapoor1" target="_blank">[45]</a>. * 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.</p