The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.
SummaryTo study the difference in expression of the chaperonin a-and b-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The b-subunit was significantly more abundant with increasing temperature (maximum at 938C), whereas the a-subunit was not. Native PAGE with Western blot analysis indicated that the natural chaperonins in the crude extracts of T. KS-1 cells grown between 658C and 958C migrate as single bands with different mobility. The recombinant a-and b-subunit homo-oligomers migrated differently from each other and from natural chaperonins. Immunoprecipitation also showed that the natural chaperonin was the hetero-oligomer. These results indicate that chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the b-subunit may be adapted to a higher temperature than the asubunit. T. KS-1 probably changes its chaperonin subunit composition to acclimatize to the ambient temperature.
Two families of FK506 binding protein (FKBP) type peptidyl-prolyl cis±trans isomerase (PPIase) have been found in Archaea. One is the 16±18 kDa short type FKBP family, and another is the 26±30 kDa long type FKBP family. The latter has a longer C-terminal region than the former. In this study, the 28.3 kDa long type FKBP gene from a thermophilic archaeum, Methanobacterium thermoautotrophicum, was expressed in Escherichia coli, and its gene product (MbFK) was characterized. The PPIase activity of MbFK was much lower than those of other FKBPs reported against oligopeptidyl substrates. MbFK protected green fluorescent protein (GFP) and rhodanese from thermal denaturation. Furthermore, MbFK suppressed the aggregation of chemically unfolded rhodanese and elevated the yield of its refolding although this activity was weaker than that of GroEL / ES. We made two deletion mutants, MbFK-N which lacked the C-terminal region, and MbFK-C which had only the C-terminal region. Far-UV CD spectra of these mutants showed that their secondary structures did not change from that of the wild-type. Whereas the PPIase activity of MbFK-N was low but detectable, that of MbFK-C was undetectable. The MbFK-C protected the thermal protein aggregation, and possessed a weak but significant aggregation suppressing activity against chemically unfolded protein. However, the MbFK-N did not suppress the aggregation of chemically unfolded rhodanese while it protected heat induced aggregation of rhodanese. These results may indicate that aggregation suppressing activity of MbFK-W against chemically unfolded protein are exerted mainly by its C-terminal domain while both domains contribute to thermal protein aggregation suppression.
Bioconversion of various substituted naphthalenes that contain 1-methoxy- and 1-ethoxy-naphthalenes, methylnaphthalenes, dimethylnaphthalenes, and naphthalenecarboxylic acid methyl esters were performed using recombinant Escherichia coli cells, which expressed the gene coding for a cytochrome P450 BM3 variant F87V (P450 BM3 (F87V)) that was N-terminally fused to an archaeal peptidyl-prolyl cis-trans isomerase. In addition, bioconversion experiments with the same substrates were carried out using those that expressed the phnA1A2A3A4 genes for a polycyclic aromatic hydrocarbon (PAH)-dihydroxylating dioxygenase, which originated from a PAH-utilizing marine bacterium Cycloclasticus sp. strain A5. Consequently, a variety of mono-hydroxylated derivatives were generated from these substituted naphthalenes. Oxidative aryl coupling was found to produce a novel compound 4,4'-diethoxy-[2,2']-binaphthalenyl-1,1'-diol from 1-ethoxynaphthalene with the E. coli cells expressing the P450 BM3 (F87V) gene. This recombinant E. coli was further shown to introduce the hydroxyl group regio- and stereo-specifically into a sesquiterpene β-eudesmol.
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FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins
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