PENICILLIN acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode1. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively2,3) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm4 and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains5. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine6'7 inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme7, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own α-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.

Penicillin acylase has a single-amino-acid catalytic centre.

http://science.sciencemag.org/content/sci/262/5132/380.full.pdf

Conformational Flexibility of Enzyme Active Sites

The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.

Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.

Inactivation precedes overall molecular conformation changes during enzyme denaturation

The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the β-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the α subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb19207.x

Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105

The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by near- and far-ultraviolet circular dichroism, steady-state and time-resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid-point of 4.5 M urea. Separation of its constituent alpha and beta peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated beta peptide aggregates over a wide range of urea concentrations but that the alpha peptide refolds reversibly to a compact state. Physical studies showed that the refolded alpha peptide has a compact but asymmetric structure with more alpha helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8-anilino-1-naphthalene sulfonic acid binding and which is normally covered by the beta peptide in the native enzyme. The results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb19206.x

The folding and solution conformation of penicillin G acylase.

Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F-inactivated chymotrypsin [Gold, A. M. & Fahrney, D. (1964) Biochemistry 3, 783–791]. Incubation of the PhMeSO2F-inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol-enzyme during incubation with 10 mM 6-aminopenicillanic acid. 4-Pyridyl-ethylcysteine was released by acid hydrolysis after reaction of the thiol-protein with 4-vinylpyridine. The rates of reaction of thiol-penicillin acylase with iodoacetic acid and 2,2′-dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol-enzyme with iodo[2-3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the β-chain NH2-terminal residue, indicating conversion of Ser290 to S-carboxymethyl-cysteine. Near-ultraviolet CD spectra showed the conformation of thiol-penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1991.tb15884.x

Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity.

The in vitro folding and assembly of penicillin acylase (EC 3.5.1.11) (PA) to active enzyme has been studied. PA is a large bacterial protein (Mr = 86,000) comprising two peptides, alpha and beta, produced by proteolytic processing and activation of a 92-kDa precursor. Proteins that result from proteolytic processing are characteristically difficult if not impossible to refold. Different factors that affect folding and assembly of PA, including pH, ionic strength, and temperature, have been studied. Yields of 60% can be obtained, based on recovery of enzyme activity, together with another 20% of folded and associated monomer with conformation closely similar to that of the active enzyme but with the active site not formed. Evidence is presented for in vitro assembly proceeding via initial folding of the N-terminal alpha-peptide with subsequent collapse of the transiently folded beta-chain on to the surface of the former. A slow process of rearrangement follows association in vitro. Competition experiments support the proposal that the linker endopeptide in the precursor serves to increase the probability of productive collision between folded alpha- and beta-peptides. The effect of raised temperature is to interfere with the folding of the alpha-peptide, thus preventing proper folding of the precursor. This finding accounts for the basis of the temperature regulation of PA production in vivo.

Christopher D. Lindsay

Papers

The folding and solution conformation of penicillin G acylase.

Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity.

Refolding and assembly of penicillin acylase, an enzyme composed of two polypeptide chains that result from proteolytic activation.