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First structural studies of dipeptidyl peptidase III from bacterial organisms

Dipeptidyl peptidase III (DPP III) is a zinc peptidase from the M49 family, widely distributed amongst eukaryotic and prokaryotic organisms. Most studies on DPP III accomplished up to now include proteins from eukaryotic organisms and show that DPP III is involved in intracellular peptide catabolism with a postulated role in pain modulation [1]. On the other hand, the knowledge about prokaryotic DPP III, its catalytic mechanism and physiological function, is very scarce. In the Protein data bank (PDB) there is no structural information of any prokaryotic DPP III. We have been studying two bacterial orthologs: DPP III from the human gut symbiont Bacteroides thetaiotaomicron (Bt) and Caldithrix abyssi (Ca), which inhabits hydrothermal vents. Both orthologs were cloned and heterologously expressed in Escherichia coli and then purified using affinity and size exclusion chromatography [2]. Crystallization screens were preformed and crystals of wild type CaDPP III were obtained. Probably due to charge heterogeneity, crystallization experiments with wild-type BtDPP III failed. In order to avoid these heterogeneities, specific amino acid replacements (all cysteines were replaced by serines) were introduced and crystals of the variant protein were obtained. Datasets for both DPP III proteins were collected on BL14.1 operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring (Berlin-Adlershof, Germany). Unfortunately, anomalous scattering of zinc was too weak for the structure to be solved using the single-wavelength anomalous dispersion of zinc atoms. Up to date, all efforts to solve Bt and Ca structures using molecular replacement method were also unsuccessful. Human [1] and yeast [3] DPP III enzymes were used as models, since their crystal structures are available. However, they have low sequence identity with the studied enzymes (sequence identity of Bt with human and yeast is 20.6 % and 17.4 %, respectively and that of Ca is 16.0 % and 14.4 %, respectively). Currently, we are preparing selenomethionine labeled protein (BtDPP III) which should help us to solve the first bacterial DPP III structure.

Dipeptidyl peptidase III ; crystallization ; X-ray diffraction

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