Bacterial β-peptidyl aminopeptidases with unique substrate specificities for β-oligopeptides and mixed β,α-oligopeptides.

We previously discovered that BapA, a bacterial β-peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert β-peptides [Geueke B, Namoto K, Seebach D & Kohler H-PE (2005) J Bacteriol 187, 5910–5917]. Here, we describe the purification and characterization of two distinct bacterial β-peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (αβ)₄ quaternary structure, the preproteins needed to be cleaved into two subunits. The two β-peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of β-peptides and mixed β/α-peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a β-amino acid at the N-terminus; peptide substrates with an N-terminal α-amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N-terminal β-amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H-βhXaa-Ile-βhTyr-OH according to the following preferences with regard to the side chain of the N-terminal β-amino acid: aliphatic and aromatic > OH-containing > hydrogen, basic and polar. Experiments with the tripeptides H-d-βhVal-Ile-βhTyr-OH and H-βhVal-Ile-βhTyr-OH demonstrated that the two BapA enzymes preferred the peptide with thel-configuration of the N-terminal β-homovaline residue as a substrate. [ABSTRACT FROM AUTHOR]