Cloning and functional expression of dipeptidyl peptidase IV from the ruminal bacterium Prevotella albensis M384(T).
Ruminal bacteria of the genus Prevotella play 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 from Prevotella albensis M384(T) was 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 in Escherichia 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 in P. albensis and, 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 of Prevotella spp. were also identified. Construction of a phylogenetic tree demonstrated that the DPP-IV of P. albensis clusters with other DPP-IVs found in bacteria of the Cytophaga-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.
Walker
Nicola D
ND
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK.
McEwan
Neil R
NR
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK.
Wallace
R John
RJ
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010802:Phylogeny; D018720:Prevotella / Q000201:enzymology* / Q000235:genetics*; D012417:Rumen / Q000382:microbiology; D017386:Sequence Homology, Amino Acid
D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D018819:Dipeptidyl Peptidase 4