Penicillin acylase has a single-amino-acid catalytic centre

Abstract

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 mode. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains. 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 cysteine inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme, 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 alpha-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.

Identification of a novel amidase motif in neutral ceramidase

“…Indeed other variations on the classical catalytic-triad theme also exist. For instance, penicillin acylase, although not a protease, hydrolyses an amide bond in substrates such as penicillin G, and it seems to use just one residue, the N-terminal serine residue, as its nucleophile [39]. Taken together, our findings imply that the neutral CDase motif is distinct from that of serine proteases and, therefore, indicate that the hexapeptide consensus sequence described here is indeed a novel amidase motif.…”

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

“…The long distances between the second cleavage sites and the Ntn Ser residues were also observed in the respective crystal structures (7,41). Although it was not confirmed, Flavobacterium glycosylasparaginase (GA) (42) may also contain an 8-aa spacer (deduced from the crystal structure available, PDB code 2gl9).…”

“…Ntn is a typical general acid-base catalytic mechanism involved in substrate hydrolysis, where the N-terminal amine group plays a role as base to deprotonate the hydroxyl group at the same residue (7). A triad charge relay system consisting of Ser 170 , His 192 , and Glu 624 was observed in CA, but this was only responsible for the first cleavage during the enzyme activation (48).…”

“…CA hydrolyzes Gl-7-ACA to 7-ACA (glutaryl 7-aminocephalosporanic acid to 7-aminocephalosporanic acid), which is a key intermediate for the synthesis of cephem antibiotics (4). Ntn hydrolases feature an ␣␤␤␣ sandwich and an N-terminal, catalytically active, nucleophile that is released by the first proteolytic step (5)(6)(7). Examples of Ntn hydrolases are glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (8), proteasome ␤-subunits (9), penicillin acylases (7), glucosamine-6-phosphate synthase (10), isoaspartyl aminopeptidase (11), taspase 1 (12), glycosylasparaginase (GA) (13,14), the human asparaginase-like protein 1 (hASRGL1) (15), Helicobacter pylori ␥-glutamyltranspeptidase (16), N-acylhomoserine lactone acylase PvdQ (PvdQ) (17), and cephalosporin acylases (18) whose crystal structures are available.…”

Improving selectivity and productivity of the enzymatic synthesis of ampicillin with immobilized penicillin G acylase

“…The amine group of serine B1 (the active site of PGA, Duggleby et al, 1995) should be uncharged, ready to accept protons when directing the nucleophilic attack on the ester binding of the substrate (PGME); thus, decreasing pH would hinder the formation of the acylenzyme complex. Besides that, at a slightly acid pH almost all the 6 -APA amine groups are still deprotonated (pK a = 2.5 and pK b = 4.9), which facilitates the nucleophilic attack on the acyl-enzyme complex by 6-APA.…”