title: A Mn(II)-Mn(II) center in human prolidase.
abstract: Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)-Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.
authors.affiliation: Department of Molecular Medicine, Section of Biochemistry, University of Pavia, via Taramelli 3 b-27100 Pavia, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Besio
authors.forename: Maria Camilla
authors.initials: MC
authors.lastname: Baratto
authors.forename: Roberta
authors.initials: R
authors.lastname: Gioia
authors.forename: Enrico
authors.initials: E
authors.lastname: Monzani
authors.forename: Stefania
authors.initials: S
authors.lastname: Nicolis
authors.forename: Lucia
authors.initials: L
authors.lastname: Cucca
authors.forename: Antonella
authors.initials: A
authors.lastname: Profumo
authors.forename: Luigi
authors.initials: L
authors.lastname: Casella
authors.forename: Riccardo
authors.initials: R
authors.lastname: Basosi
authors.forename: Ruggero
authors.initials: R
authors.lastname: Tenni
authors.forename: Antonio
authors.initials: A
authors.lastname: Rossi
authors.forename: Antonella
authors.initials: A
authors.lastname: Forlino
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D004150:Dipeptidases / Q000737:chemistry* / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
chemical_list: D004792:Enzyme Precursors; D008345:Manganese; D004150:Dipeptidases; C022449:proline dipeptidase