An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity.
The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cis/trans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatically active fragment could be isolated after proteolysis by subtilisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni2+-NTA column. Subsequent digestion with subtilisin and anion-exchange chromatography yielded a TF fragment encompassing amino acids Gln-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 microM(-1) s(-1) could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrolide FK506.
Stoller
G
G
Max-Planck-Gesellschaft, Arbeitsgruppe Enzymologie der Peptidbindung, Halle/S., Germany.
Tradler
T
T
Rücknagel
K P
KP
Rahfeld J-U
Fischer
G
G
D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry*; D019696:Peptidylprolyl Isomerase; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013381:Subtilisins / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013381:Subtilisins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus