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 the rmophilic methanogen, Methanococcus thermolithotrophicus. The PPIase a ctivity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodec yl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtra tion. The enzyme was thermostable, with the half-lives of its activity at 90 and 100 degrees C being 90 and 30 min, respectively. The cataly tic efficiencies (k(cat)/K-m) measured at 15 degrees C for the peptidy l substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl -Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 mu M-1 s(-1), resp ectively, in chymotrypsin-coupled assays. The purified enzyme was sens itive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. t hermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed th at MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermost able nature of MTFK and its structure is discussed.