Autoproteolytic and Catalytic Mechanisms for the β-Aminopeptidase BapA—A Member of the Ntn Hydrolase Family

Abstract

The β-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal β-amino acid residues from peptides. We determined the crystal structures of the native (αβ)₄ heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.

Crystal Structures of BapA Complexes with β‐Lactam‐Derived Inhibitors Illustrate Substrate Specificity and Enantioselectivity of β‐Aminopeptidases

“…Leu84, Val88, Gln90, Leu92, Phe124, Ser125, Leu127, Leu128, and Leu303) constitute a hydrophobic pocket that supports ligand binding by van der Waals interactions ( Figure 1). The aromatic ring of Phe124, which showed very weak electron density in the native structure of BapA, [18] is well defined through a p-stacking interaction with the aromatic ring of AEBSF.…”

“…ability to process b-peptides is not restricted to the few described b-aminopeptidases. [18] The large abundance of b-aminopeptidase-like sequences suggests that these enzymes have important cellular functions that are as yet unknown. Although peptides composed solely of linear b-amino-acid residues do not occur in nature, many b-amino acids are constituents of a wide variety of different, often highly bioactive substances, such as microcystins, [19,20] taxol, [21] bestatin, [22,23] and l-carnosine.…”

“…Furthermore, the precursor structure of BapA and an analysis of active-site mutants revealed that self-processing of BapA is unlikely to follow the classical autoproteolytic mechanism of Ntn hydrolases but instead involves different nucleophiles for self-processing and catalysis. [18] On the basis of the available crystal structures, it has been suggested that the topological and functional properties of Ntn hydrolases and b-aminopeptidases resulted from convergent evolution of different evolutionary origins. [38] Based on point mutations at the BapA active site, we have proposed a mechanism of the BapA-catalyzed reaction.…”

“…[38] Based on point mutations at the BapA active site, we have proposed a mechanism of the BapA-catalyzed reaction. [18] Essential functions were assigned to the N-terminal catalytic nucleophile Ser250, which is activated by the Ser288/Glu290 dyad. However, the molecular determinants of the unusual bpeptide specificity of b-aminopeptidases have not been investigated in detail, as all attempts to obtain crystal structures of b-aminopeptidase-ligand complexes and to identify b-aminopeptidase-specific inhibitors have as yet been unsuccessful.…”

Exploring the Role of Conformational Heterogeneity in cis-Autoproteolytic Activation of ThnT

“…The Nuc → Ala variant of β-aminopeptidase (BapA) crystallized in a conformation comparable to the inactive state of ThnT. 54 From these data, it was proposed that BapA did not undergo the canonical N → O acyl shift mechanism, but rather an unprecedented trans -type autoproteolysis. This proposal, while provocative, lacked the discriminating experiment in which the Nuc → Cys variant is chemically rescued by addition of hydroxylamine.…”