Abstract
Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family.
Highlights
Bacteroides thetaiotaomicron is one of the best studied representatives of the Bacteroides spp, a group of anaerobic, Gram-negative microbes, which are part of the human intestinal microbiome and play a central role in symbiotic host-bacterial relationships in the human gut [1]
We propose that dipeptidyl peptidase III (DPP III) is involved in protein metabolism in B. thetaiotaomicron and in many other Bacteroides species and that it contributes to the growth of these bacteria
Our results revealed that the overall protein fold is very similar to that of the human and yeast orthologue, with two domains separated by a wide cleft containing a catalytic zinc ion
Summary
Bacteroides thetaiotaomicron is one of the best studied representatives of the Bacteroides spp, a group of anaerobic, Gram-negative microbes, which are part of the human intestinal microbiome and play a central role in symbiotic host-bacterial relationships in the human gut [1]. These bacteria are known for their ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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