PMID:15843029

Citation	Ignatova, Z, Wischnewski, F, Notbohm, H and Kasche, V (2005) Pro-sequence and Ca2+-binding: implications for folding and maturation of Ntn-hydrolase penicillin amidase from E. coli. J. Mol. Biol. 348:999-1014
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.
Links	PubMed Online version:10.1016/j.jmb.2005.03.005
Keywords	Amidohydrolases/chemistry; Amidohydrolases/genetics; Amidohydrolases/metabolism; Amino Acid Sequence; Binding Sites; Calcium/metabolism; Calcium/pharmacology; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Kinetics; Models, Molecular; Molecular Sequence Data; Penicillin Amidase/chemistry; Penicillin Amidase/genetics; Penicillin Amidase/metabolism; Protein Binding; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Sequence Alignment; Thermodynamics; Urea/pharmacology
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