title: Biochemical characterization of deblocking aminopeptidases from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.
abstract: Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.
authors.affiliation: College of Plant Sciences, Jilin University, Changchun, China.
authors.forename: Baolei
authors.initials: B
authors.lastname: Jia
authors.forename: Sangmin
authors.initials: S
authors.lastname: Lee
authors.forename: Bang Phuong
authors.initials: BP
authors.lastname: Pham
authors.forename: Jae Myeng
authors.initials: JM
authors.lastname: Kwack
authors.forename: Haifeng
authors.initials: H
authors.lastname: Jin
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Yuhan
authors.initials: Y
authors.lastname: Wang
authors.forename: Gang-Won
authors.initials: GW
authors.lastname: Cheong
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D015870:Gene Expression; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010957:Plasmids; D058105:Polymerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D019843:Archaeal Proteins; D007527:Isoenzymes; D009842:Oligopeptides; D011994:Recombinant Proteins; C039944:alanyl-alanyl-alanine; D000626:Aminopeptidases