title: Pro-sequence and Ca2+-binding: implications for folding and maturation of Ntn-hydrolase penicillin amidase from E. coli.
abstract: Penicillin amidase (PA) is a bacterial periplasmic enzyme synthesized as a pre-pro-PA precursor. The pre-sequence mediates membrane translocation. The intramolecular pro-sequence is expressed along with the A and B chains but is rapidly removed in an autocatalytic manner. In extensive studies we show here that the pro-peptide is required for the correct folding of PA. Pro-PA and PA unfold via a biphasic transition that is more pronounced in the case of PA. According to size-exclusion chromatography and limited proteolysis experiments, the inflection observed in the equilibrium unfolding curves corresponds to an intermediate in which the N-terminal domain (A-chain) still possesses native-like topology, whereas the B-chain is unfolded to a large extent. In a series of in vitro experiments with a slow processing mutant pro-PA, we show that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude. In the absence of the pro-domain the PA refolds to a stable inactive molten globule intermediate that has native-like secondary but little tertiary structure. The pro-sequence of the homologous Alcaligenes faecalis PA can facilitate the folding of the hydrolase domain of Escherichia coli PA when added in trans (as a separate polypeptide chain). The isolated pro-sequence has a random structure in solution. However, difference circular dichroism spectra of native PA and native PA with pro-peptide added in trans suggest that the pro-sequence adopts an alpha-helical conformation in the context of the mature PA molecule. Furthermore, our results establish that Ca2+, found in the crystal structure, is not directly involved in the folding process. The cation shifts the equilibrium towards the native state and facilitates the autocatalytic processing of the pro-peptide.
authors.affiliation: Institute of Biotechnology II, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany. ignatova@tu-harburg.de
authors.forename: Zoya
authors.initials: Z
authors.lastname: Ignatova
authors.forename: Frank
authors.initials: F
authors.lastname: Wischnewski
authors.forename: Holger
authors.initials: H
authors.lastname: Notbohm
authors.forename: Volker
authors.initials: V
authors.lastname: Kasche
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D014508:Urea; D000581:Amidohydrolases; C438834:N-terminal nucleophile hydrolase; D010399:Penicillin Amidase; D002118:Calcium