Cloning and functional expression of dipeptidyl peptidase IV from the ruminal bacterium Prevotella albensis M384T

Abstract

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.

Cloning, Purification, and Enzymatic Properties of Dipeptidyl Peptidase IV from the Swine Pathogen Streptococcus suis

“…The substrate showing the highest affinity for the recombinant DPP IV was Gly-Pro-pNa, with a K m of 0.26 mM and a V max of 7.8 mol mg Ϫ1 min Ϫ1 . This value is comparable to those reported for P. albensis (27) and L. helveticus (26). The recombinant DPP IV also had significant affinity for Lys-AlapNa (K m of 0.87 mM) and Ala-Pro-pNa (K m of 12 mM) with V max values of 5.9 and 30 mol mg Ϫ1 min Ϫ1 , respectively.…”

“…The deduced amino acid sequence from the nucleotide sequence revealed that it contained a complete open reading frame coding for a 755-aminoacid protein with a calculated molecular mass of 85 kDa. This correlates well with the molecular masses previously reported for other DPP IVs, including those from P. albensis (27), S. gordonii (7), Lactobacillus sakei (20), and S. thermophilus (25).…”

“…Several incubation temperatures were tested (4, 10, 24, 37, 45, and 55°C) using PBS as the buffer and the optimal temperature was found to be 37°C (Table 4). These optimal conditions observed for the S. suis recombinant DPP IV were similar or close to those reported for other bacterial DPP IVs (2,7,17,25,27). To evaluate the stability of the recombinant DPP IV, the enzyme was diluted to the working concentration and kept for 24 h at Ϫ20, 4, 10, or 24°C prior to assaying the activity as described above (Table 4).…”

“…No differences were noted for extracellular DPP IV activity. The modulation of DPP IV by casein hydrolysate was also observed for P. albensis (27). The glucose concentration had no effect on either the cell-associated or extracellular DPP IV activity of S. suis S735.…”

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

“…In the rumen they are symbionts contributing to the degradation of plant cell-wall polysaccharides (Miyazaki et al, 2003) and protein metabolism (Walker et al, 2003), whereas oral prevotellas are implicated in dental plaque establishment (Kolenbrander et al, 2002), periodontitis (Paster et al, 2001), advanced caries (Martin et al, 2002), oro-pharyngeal abscesses (Brook, 2004) and noma, a grotesque childhood orofacial gangrene, now confined mostly to sub-Saharan Africa (Enwonwu et al, 2006). The genetic tools that would make possible more thorough studies of prevotellas are still undeveloped despite their ecological and medical importance.…”