Cloning and functional expression of dipeptidyl peptidase IV from the ruminal bacterium Prevotella albensis M384T
Open Access
- 1 August 2003
- journal article
- Published by Microbiology Society in Microbiology
- Vol. 149 (8), 2227-2234
- https://doi.org/10.1099/mic.0.26119-0
Abstract
Ruminal bacteria of the genusPrevotellaplay a crucial role in peptide breakdown in the rumen, a component of protein catabolism that leads to the inefficient use of dietary protein by ruminant animals. This is the first report of the cloning of a peptidase gene from a ruminal bacterium. Part of the dipeptidyl peptidase type IV (DPP-IV) gene fromPrevotella albensisM384Twas cloned using degenerate primers designed from conserved regions found within other known DPP-IV sequences. Flanking regions were determined by genomic walking. The DPP-IV gene was expressed inEscherichia coli. The cloned enzyme required a free N terminus and catalysed the removal of X-Pro dipeptide from proline-containing oligopeptides, where proline was the second residue from the N terminus. It was inhibited by serine protease inhibitors and the substrate analogue for mammalian DPP-IV, diprotin A. The properties of the cloned enzyme were similar to those of the native form inP. albensisand, in general, DPP-IVs from other organisms. The enzyme contained a conserved motif which is associated with the S9 class of prolyl oligopeptidases. The DPP-IV gene appeared not to be part of a contiguous operon. Regions with similarity to other putative promoters ofPrevotellaspp. were also identified. Construction of a phylogenetic tree demonstrated that the DPP-IV ofP. albensisclusters with other DPP-IVs found in bacteria of theCytophaga–Flexibacter–Bacteroidaceae(CFB) phylum, which are more closely related to eukaryotic DPP-IVs than the DPP-IV-like enzyme (PepX) of the lactic acid bacteria.Keywords
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