Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolylcis-transIsomerase from a Thermophilic Archaeon,Methanococcus thermolithotrophicus

Abstract

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°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat /K m ) measured at 15°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 M ؊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.

The 28.3 kDa FK506 binding protein from a thermophilic archaeum, Methanobacterium thermoautotrophicum, protects the denaturation of proteins in vitro

“…MbFK-N seemed to form a dimer in a similar fashion to the short type archaeal FKBPs from Mc. thermolithotrophicus [17] and Thermococcus sp. KS-1 [18].…”

“…The N-terminal domains of the long type archaeal FKBPs have the archaea specific insertions, in the regions corresponding to`bulge' and flap' of hFKBP12, which are also present in the short type archaeal FKBPs (Fig. 1A) [17]. MbFK-W, and its deletion mutants, MbFK-N and MbFK-C were expressed (Fig.…”

“…thermolithotrophicus and Thermococcus sp. KS-1 which were reported as 42 kDa [17] and 30 kDa [18], respectively ( Table 1). The apparent molecular masses of MbFK-W, MbFK-N and MbFK-C obtained by gel filtration chromatography were 5.7-, 1.9-and 6.0-fold of their theoretical values.…”

“…The apparent molecular masses of MbFK-W and its deletion mutants were estimated by gel filtration chromatography using a TSKgel G3000 SW XL column. The theoretical molecular masses were calculated from reported amino-acid sequences [17,18,25]. Aggregation suppressive activities of the MbFK mutants against chemically unfolded rhodanese…”

“…We have previously reported two archaeal FKBPs, one from thermophilic methanogen, Methanococcus thermolithotrophicus (MTFK) [17] and another from a hyperthermophilic archaeum, Thermococcus sp. KS-1 (TcFK) [18].…”