Structural characterization of dipeptidyl peptidase III from the bacterium Bacteroides thetaiotaomicron

Abstract

Dipeptidil-peptidaza III (DPP III) je široko rasprostranjena citosolna metalopeptidaza iz porodice M49, koja odcjepljuje dipeptide s N-kraja peptidnih supstrata. Većina istraživanja o DPP III napravljena je na ortolozima iz eukariota koja ukazuju na njenu opću funkciju u intracelularnom katabolizmu proteina, ulogu u regulaciji boli te odgovoru na oksidativni stres. U ovom radu je strukturno karakterizirana prva prokariotska DPP III, i to iz bakterije Bacteroides thetaiotaomicron, ljudskog simbionta koji nastanjuje tanko i debelo crijevo. Kinetičkom analizom utvrđeno je da je Arg2-2-naftilamid njezin najbolji sintetski supstrat. Elektroforetskim metodama je utvrđeno da pročišćeni proteinski uzorak sadrži tri forme DPP III. Zamjenom svih cisteinskih ostataka u serinske pripremljen je dovoljno homogen uzorak za kristalizaciju bakterijske DPP III. Riješene su strukture u otvorenoj i zatvorenoj konformaciji proteina. Pokazalo se da ovaj protein pripada istoj strukturnoj klasi kao i ljudska i kvaščeva DPP III. S obzirom na gotovo jednako aktivno mjesto može se pretpostaviti i isti mehanizam djelovanja.Dipeptidyl peptidase III (DPP III) is a widespread cytosolic metalopeptidase of the M49 family, which cleaves dipeptides from the N-terminus of its peptide substrates. Previous studies of the DPP III enzyme were focused on eukaryotic orthologs and indicate its general function in the intracellular protein catabolism, pain regulation as well as the response to oxidative stress. In this work, the first prokaryotic DPP III, that from the human gut symbiont bacterium Bacteroides thetaiotaomicron, was structurally characterized. Kinetic analysis showed that Arg2-2-naphthylamide is the preferred synthetic substrate. Electrophoretic methods revealed that the purified protein sample contains three forms. By replacing all cysteine residues with serines, homogeneity of the sample improved enough to enable successful crystallization of the bacterial DPP III. Structures in open and closed conformations were solved and they have the same protein fold as the human and yeast DPP III. Considering the fact that these three enzymes have almost identical active sites, the same mechanism of action can be assumed