title: An archaeal protein evolutionarily conserved in prokaryotes is a zinc-dependent metalloprotease.
abstract: A putative protease gene (tldD) was previously identified from studying tolerance of letD encoding the CcdB toxin of a toxin-antidote system of the F plasmid in Escherichia coli. While this gene is evolutionarily conserved in archaea and bacteria, the proteolytic activity of encoded proteins remained to be demonstrated experimentally. Here we studied Sso0660, an archaeal TldD homologue encoded in Sulfolobus solfataricus by overexpression of the recombinant protein and characterization of the purified enzyme. We found that the enzyme is active in degrading azocasein and FITC-BSA substrates. Protease inhibitor studies showed that EDTA and o-phenanthroline, two well-known metalloprotease inhibitors, either abolished completely or strongly inhibited the enzyme activity, and flame spectrometric analysis showed that a zinc ion is a cofactor of the protease. Furthermore, the protein forms disulfide bond via the Cys416 residue, yielding protein dimer that is the active form of the enzyme. These results establish for the first time that tidD genes encode zinc-containing proteases, classifying them as a family in the metalloprotease class.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Hu
authors.forename: Nan
authors.initials: N
authors.lastname: Peng
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Han
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Mei
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Chen
authors.forename: Xu
authors.initials: X
authors.lastname: Feng
authors.forename: Yun Xiang
authors.initials: YX
authors.lastname: Liang
authors.forename: Qunxin
authors.initials: Q
authors.lastname: She
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012710:Serum Albumin, Bovine / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D002364:Caseins; D011994:Recombinant Proteins; C026018:azocasein; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D045726:Metalloproteases; D016650:Fluorescein-5-isothiocyanate; D015032:Zinc