β‐Aminopeptidase‐Catalyzed Biotransformations of β²‐Dipeptides: Kinetic Resolution and Enzymatic Coupling

Abstract

We have previously shown that the beta-aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non-natural beta(3)-peptides and beta(3)-amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N-terminal beta(2)-amino acid residues as substrates under aqueous conditions. The suitability of a beta(2)-peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C(alpha) substituent of the N-terminal beta(2)-amino acid. BapA was shown to convert a diastereomeric mixture of the beta(2)-peptide H-beta(2)hPhe-beta(2)hAla-OH, but did not act on diastereomerically pure beta(2),beta(3)-dipeptides containing an N-terminal beta(2)-homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA-catalyzed transformation of the diastereomeric mixture of H-beta(2)hPhe-beta(2)hAla-OH proceeded along two highly S-enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis-to-hydrolysis ratio (v(S)/v(H)) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N-terminal beta-amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of beta(2)-homophenylalanine was detected (ee>99 % for H-(S)-beta(2)-hPhe-OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure beta(2)-amino acids and the enantioselective coupling of N-terminal beta(2)-amino acids to peptides.

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

“…In contrast to BapA, which exclusively cleaves peptides carrying N-terminal b-amino acids with aliphatic, aromatic, or functionalized side chains, DmpA preferentially reacts with substrates carrying unsubstituted or sterically undemanding N-terminal b-amino acids (e.g., b-homoglycine and b 3 -homoalanine) as well as aamino acids. [7,10,16,39] Superposition of DmpA on the modeled BapA complexes with AEBSF and Amp hyd perfectly illustrates this steric restriction of the DmpA active site ( Figure 5). Although BapA has a rather open ligand-binding pocket, the active site of DmpA is largely occluded by a loop ranging from Gln131 to Trp137; in particular, the side chain of Trp137 protrudes far towards the catalytic nucleophile Ser250 and could therfore 1) force substrates with N-terminal a-amino acids into a position that allows for nucleophilic attack of Ser250-Og, and 2) restrict catalysis by DmpA to substrates with sterically undemanding side chains.…”

“…b 2 -Dipeptides with small and therefore less demanding side chains at the N-terminal aminoacid residue are not converted by BapA, irrespective of their configuration. [16] As suggested for the conversion of substrates carrying N-terminal d-b 3 -aminoacid residues with side chains of different sizes, the preference of BapA for bulky b 2 -amino-acid residues may likewise be explained by the stabilization of the enzyme-substrate complex by increased hydrophobic interactions between large amino acid side chains and the ligand binding pocket.…”

“…In addition to its high catalytic activity with substrates composed of l-b 3 -amino acids, BapA also slowly processes a diastereomeric mixture of the b 2 -dipeptide H-b 2 hPhe-b 2 hAla-OH with high d-enantioselectivity. [16] In order to elucidate the structural details of this enantioselective reaction, we modeled the reaction intermediates formed by the nucleophilic addition of BapA Ser250-Og to the carbonyl group of the (+)-sc conformers of d-and l-b 2 -homophenylalanine residues ( Figure S2). Due to the previously mentioned flexibility of the Phe124 side chain, the tetrahedral reaction intermediate of d-b 2 -homophenylalanine could be placed in the active site of BapA.…”

“…[12] The growing demand for b-amino-acid-based building blocks [13] has led to the development of biocatalytic applications for b-aminopeptidases, such as the preparation of enantiopure b-amino acids by kinetic resolution and the preparation of band mixed b,a-peptides from mildly activated bamino acids. [14][15][16][17] Alignment searches revealed many sequences from bacterial and eukaryotic genomes that have similarity with the known b-aminopeptidases. [12] Within these sequences, the catalytically relevant amino acids are highly conserved, indicating that the b-Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of b-amino acids, which serve as building blocks for the design of non-natural peptidomimetics.…”

Hydrolysis of Amides

“…The two Sphingosinicella BapA aminopeptidases, in contrast, showed much broader substrate specificity; all four of the tested b 3 -amino acid amides were converted with acceptable rates (0.38-16 U mg À1 protein) and moderate to excellent enantioselectivities (E > 53). Given that very recently the application of DmpA and 3-2W4 BapA for the synthesis of enantiopure b 2 -amino acids has also been published [334], these b-aminopeptidases form a new and promising enzyme platform for the production of a wide array of enantiopure b-amino acids under mild conditions.…”