?-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate.

ABSTRACT: ?-Aminopeptidases have the unique capability to hydrolyze N-terminal ?-amino acids, with varied preferences for the nature of ?-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of ?-peptides and to kinetically resolve ?-peptides and amides for the production of enantiopure ?-amino acids. To date, six ?-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus Here we report on the purification and characterization of an additional four ?-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding ?-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The ?-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated ? and ?, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the ?-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations ?-homo-Gly (?hGly) and ?3-homo-Leu (?3hLeu) with high activities. These enzymes displayed higher activity with H-?hGly-p-nitroanilide (H-?hGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new ?-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce ?-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the ?-aminopeptidases may play a role in some cellular functions.IMPORTANCE ?-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic ?-peptides as a sole carbon source. Six ?-aminopeptidases have been previously characterized with preferences for different ?-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic ?-peptides but also the synthesis of short ?-peptides. Identification of other ?-aminopeptidases adds to this toolbox of enzymes with differing ?-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of ?-amino acid derivatives and ?-peptides for use in biomedical and biomaterial applications. This is important, because ?-amino acids and ?-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to ?-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

SUBMITTER: John-White M 

PROVIDER: S-EPMC6643246 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature