title: Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system.
abstract: The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.
authors.affiliation: Facultad de Ciencias, Universidad de Alicante, Alicante, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Esclapez
authors.forename: B
authors.initials: B
authors.lastname: Zafrilla
authors.forename: R M
authors.initials: RM
authors.lastname: Martínez-Espinosa
authors.forename: M J
authors.initials: MJ
authors.lastname: Bonete
mesh_terms: D000595:Amino Acid Sequence; D015756:Genes, tat; D019623:Haloferax mediterranei / Q000235:genetics* / Q000378:metabolism*; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D017510:Protein Folding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D008667:Metalloproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing