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

Abstract

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 Bacteriol187, 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 the l-configuration of the N-terminal β-homovaline residue as a substrate. © 2006 The Authors.