Insight into the substrate length restriction of M32 carboxypeptidases: characterization of two distinct subfamilies.

M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted. Structural and sequence analyses implicate a highly conserved Arg at the back of the groove as being critical for this length restriction. However, the Thermus thermophilus and Bacillus subtilis M32 members lack this conserved Arg. Herein, we present the biochemical and structural characterization of these two proteins. Our findings support the important role of the conserved Arg in maintaining the length restriction, and reveal a proline-rich loop as an alternate blocking strategy. Based on our results, we propose that M32 carboxypeptidases from Bacilli belong to a separate subfamily.

A proposed role for Leishmania major carboxypeptidase in peptide catabolism.

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.