Biochemical and genetic characterization of an FK506-sensitive peptidyl prolyl cis-trans isomerase from a thermophilic archaeon, Methanococcus thermolithotrophicus.
A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100 degrees C being 90 and 30 min, respectively. The catalytic efficiencies (k(cat)/Km) measured at 15 degrees C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 microM(-1) s(-1), respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.
Furutani
M
M
Marine Biotechnology Institute, Kamaishi Laboratories, Iwate, Japan. mfurutani@kamaishi.mbio.co.jp
Iida
T
T
Yamano
S
S
Kamino
K
K
Maruyama
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics; D004795:Enzyme Stability; D006360:Heat-Shock Proteins / Q000235:genetics; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus