Isolation and Amino Acid Sequence of a New 22-kDa FKBP-like Peptidyl-prolyl cis/trans-Isomerase of Escherichia coli

Abstract

We identified a periplasmic peptidyl-prolyl cis/transisomerase (PPIase) of the (FK506-binding protein (FKBP) type in Escherichia coli (FK506 represents a natural peptidomacrolide containing an acylated pipecolic acid residue). After purification to homogeneity, its complete amino acid sequence was determined by a combination of Edman degradation and electrospray mass spectrometry of the authentic protein and peptides generated by proteolysis. The molecular mass calculated from the amino acid sequence of the protein was 22,085.53 Da, which corresponded perfectly with the value of 22,084 ؎ 1.47 Da as determined by mass spectrometry. The corresponding gene was cloned and analyzed, and Southern blot experiments revealed the existence of similar genes in various Gram-negative bacteria. The amino acid sequence of the novel FKBP22 shows similarity to Mip (macrophage infectivity potentiator)-like proteins produced by a number of pathogenic bacteria. However, FKBP22 is inhibited more strongly by FK506 than are other Mip-homologues, as indicated by the K i value of 25 nM. The subsite specificity regarding the P 1 position of the substrate resembles that for Mip-FKBP25 from Legionella pneumophila. The mature FKBP22 enzyme of 205 amino acids exists as a dimer in solution.The observation of an accelerated cis/trans isomerization of the oligopeptide succinyl-Ala-Ala-Pro-Phe-4-nitroanilide in biological material led to the discovery of peptidyl-prolyl cis/ trans-isomerases (PPIases, E.C. 5.2.1.8) 1 in 1984 (1). Recently, it was shown that they can also act on polypeptides as folding helper enzymes during the refolding of proteins in vitro (2) and in vivo (3,4). By comparison of their amino acid sequences PPIases can be subdivided into three families: the cyclophilins (5), the FK506-binding proteins (FKBPs) (6), and the parvulins (7,8). The families consist of many different members even in the same cell type or organism (9, 50). Little data exist describing the occurrence of Mip-like proteins or PPIases of the FKBP family in Enterobacteriaceae such as Escherichia coli. In addition to two members of the cyclophilin family of PPIases, E. coli also contains the 10.1-kDa PPIase parvulin (7), the 48-kDa trigger factor (21), and three FKBP-like genes. The predicted protein product of one open reading frame, orf149, shows similarities to the FKBPs (22). The slyD gene (23) and its protein product (24) is 47.3% similar to the FKBP family but the protein does not exhibit PPIase activity in the standard enzyme assay. The amino acid sequence deduced from the recently discovered fkpA gene is much more related to the Mip-like FKBPs (25), showing 83% identity to the consensus sequence of the catalytic core as derived by Trandinh et al. (26). In all these cases, it remains unclear whether or not the deduced proteins have PPIase activity and contribute to the PPIase pattern of E. coli.In this study we describe the purification and characterization of a new periplasmic, Mip-like PPIase from E. coli, and show that similar genes are present in ot...

Global Genomic Analysis of AlgU (σ E )-Dependent Promoters (Sigmulon) in Pseudomonas aeruginosa and Implications for Inflammatory Processes in Cystic Fibrosis

“…However, at least one of these putative gene products most likely plays a role in protein folding. LptB shows strong homology to FKBP-like peptidylprolyl cis/trans isomerases, which catalyze the interconversion of the proline peptide bonds between the cis and trans isomers (46). This proposed function of LptB is in keeping with the nature of the majority of genes controlled by AlgU (i.e., stress response or protein folding) identified here and elsewhere (14,18,32,38).…”

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

“…Below Hsa12 are the sequences for the homologues of FKBP12 in the eukaryotes N. crassa (Ncr16) and S. cerevisiae (Sce11). The three sequences below the eukaryotic sequences are Mip sequences: the Mip protein of L. pneumoniae (LpMip) and the Mip-like sequences of E. coli [EcMip1, also known as FkpA (Horne and Young, 1995), and EcMip2, also known as FklB (Rahfeld et al, 1996)]. The bottom sequence is the E. coli trigger factor (EcTig).…”

“…The tig gene, at 9.8 min, encodes the 'trigger factor', which was originally thought to be involved in protein secretion (Crooke and Wickner, 1987) but more recently has been identified as an abundant FKBP-type PPIase associated with ribosomes and nascent peptides (Stoller et al, 1995;Hesterkamp and Bukau, 1996). Two Mip-like FKBP PPIases have been identified, one encoded by fkpA at 74.9 min (Horne and Young, 1995) and the other by fklB at 95.4 min (Rahfeld et al, 1996). slpA (formerly orf149) is part of the lspA operon at 0.6 min and encodes a cytosolic PPIase of unknown function (Bouvier and Stragier, 1991;Hottenrott et al, 1997).…”

Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of Legionella pneumophila

“…Cross-linking experiments with dimethyl pimelimidate (DMP) revealed that Legionella Mip forms homodimers both in solution and on the bacterial surface (43,44). In this respect the Legionella protein resembles FKBP22 and FkpA from E. coli, which were also shown to form dimers in solution (35,36). More recently, the 2.4-Å crystal structure of the Mip protein from L. pneumophila Philadelphia 1 was determined (39).…”