FKBP-type peptidyl-prolyl cis-trans isomerase from a sulfur-dependent hyperthermophilic archaeon, Thermococcus sp. KS-1.
A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17kDa and the other 27kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC50 of 7 microM. This enzyme showed high kinetic stability with a half-life of 40 min at 100 degrees C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.
Iida
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T
Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan.
Furutani
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M
Nishida
F
F
Maruyama
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D021983:Cyclophilins; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D006801:Humans; D020104:Immunophilins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D022021:Tacrolimus Binding Proteins; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D019641:DNA, Archaeal; D021983:Cyclophilins; C114522:PPIE protein, human; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase