Cyclophilin and trigger factor from Bacillus subtilis catalyze in vitro protein folding and are necessary for viability under starvation conditions.

Abstract

Cyclophilin (the product of the ppiB gene) and the trigger factor (the product of the tig gene) are the only cytosolic peptidyl-prolyl cis-trans isomerases that are known in Bacillus subtilis. Both enzymes catalyze the in vitro refolding of ribonuclease T1, a reaction that is limited in rate by a prolyl cis/trans isomerization. The efficiency of cyclophilin as a folding catalyst is only modest with a kcat/KM value of 3.8 x 10(4) M-1 s-1, but the trigger factor shows an almost 40-fold higher specific activity with a kcat/KM value of 1.4 x 10(6) M-1 s-1. This high catalytic activity originates from the tight binding to the protein substrate as reflected in both the low KM value of 0.5 microM and in the strong inhibition of the trigger factor by unfolded proteins. By use of a protein-folding assay, the concentrations of cyclophilin and the trigger factor in the cytosol of B. subtilis could be determined as 26 and 35 microM, respectively. Together they account for the entire folding activity that is detectable in crude extracts of wild-type B. subtilis cells. The genes encoding cyclophilin and the trigger factor in the B. subtilis chromosome were disrupted individually and simultaneously. Even in combination, these disruptions had no effect on cell viability in rich medium or under several stress conditions, such as heat, osmotic, or oxidative stress. However, in poor medium and, in particular, in the absence of amino acids, the growth of the double mutant strain was strongly decelerated, indicating that the prolyl isomerases become essential for growth under starvation conditions. It is not yet known whether this function relates to the catalysis of the proline-limited folding of essential proteins.

• Authors: S F Göthel, C Scholz, F X Schmid, M A Marahiel
• Journal: Biochemistry (Biochemistry) Vol. 37 Issue 38 Pg. 13392-9 (Sep 22 1998) ISSN: 0006-2960 [Print] United States
• PMID: 9748346 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Amino Acids
• Culture Media
• Isoenzymes
• Peptidylprolyl Isomerase

Topics

• Amino Acids (deficiency)
• Bacillus subtilis (enzymology, genetics, growth & development)
• Catalysis
• Culture Media (chemistry)
• Gene Deletion
• Genes, Bacterial (genetics)
• Isoenzymes (chemistry, genetics, metabolism)
• Peptidylprolyl Isomerase (chemistry, genetics, metabolism)
• Protein Folding