Penicillin G Acylase
Created by Gracie Brown-Geist
Penicillin G acylase (PDB ID: 1AJQ) is found in the organism Escherichia coli. Penicillin G acylase has a molecular weight of 94,642.59 Da, and its isoelectric point (pI) is 6.17. It is part of the larger family of penicillin acylases that come from such microorganisms as bacteria, yeast, and fungi. Penicillin acylases catalyze the hydrolysis of naturally occurring penicillins, due to their high specificity for the amide bond of penicillin (4). This reaction yields two products: a carboxylate (phenylacetic acid in the specific case of penicillin G acylase) and 6-aminopenicillanic acid (6-APA) (1). The reaction requires water to proceed. These enzymes fall into three subcategories based on which form of penicillin they prefer to have as a substrate. The protein discussed here falls into the group that preferentially hydrolyses penicillin G, but there is also a group that prefers penicillin V (penicillin V acylases), and a group that prefers ampicillin (ampicillin acylases) (2). While the aforementioned penicillins are the main substrates of this set of enzymes, they also have the capability of hydrolyzing a wide range of amides (they are also referred to as penicillin amidases) (2).
Penicillin G acylase is initially synthesized in an organism as a single chain precursor consisting of 846 amino acids. Its production occurs in the cytoplasm, and after processing it is transported into the periplasm, which is where it catalyzes hydrolysis (1). The processing of penicillin G acylase into a mature and enzymatically active heterodimer entails the removal of 26 residue signal peptide and a 54 residue spacer peptide (1). The signal peptide is necessary for localization into the periplasm, and the spacer peptide ensures proper folding prior to cleavage (2). Once it is correctly folded and cleaved, penicillin G acylase consists of an A chain containing 209 amino acid residues, and a B chain containing 557 amino acid residues (1). The B chain contributes all of the catalytic activity, but the A chain contributes to the formation of the binding pocket (3).The above-described processing is autocatalytic, and it has also been found through mutation analyses to be dependent upon the Arg B263 residue (2). Mutating this residue results in the accumulation of the precursor form of penicillin G acylase. During this assembly the A and B chains each direct their own folding, as each has been shown to fold when isolated (6). The two chains must associate early in the folding process, otherwise they tend to self-aggregate (6).
Penicillin G acylase is biologically significant because it possesses great pharmaceutical importance. 6-APA, one of the products of the hydrolysis reaction catalyzed by penicillin G acylase, is used to synthesize several semi-synthetic penicillin antibiotics (1). Penicillin G acylase has laboratory significance as well, as it protects amino and hydroxyl groups during the synthesis of peptides (5). Additionally, it is used in the resolution of racemic mixtures of chiral compounds (5). Although the natural function of penicillin G acylase has yet to be elucidated with confidence, it is thought to aid in the production of phenylacetic acid via the degradation of phenylacetylated compounds (1). This is significant because phenylacetic acid may be used as a carbon source for bacteria when it is in a free-living mode (1). Discovery of the presence of phenylacetic acid within a pocket of penicillin G acylase revealed this enzyme’s substrate binding site (2).
The binding site of penicillin G acylase is notable because it adopts one of two possible conformations based on the substrate it is binding. The conformation is determined by the positions of Arg A145 and Phe A146, as these are the only residues demonstrating a significant shift between the two forms (1). The existence of two energetically favorable conformations of the binding site is possible because of the kink present between the Met A142 and Ala A143 residues, which are part of a 16-residue alpha helix (2). Arg A145 and Phe A146 are found at the end of this helix, and they are able to shift because this kink destabilizes the helix (2). This potential for shifting within the binding pocket expands the list of possible ligands to include molecules with acid groups extending out of the binding pocket, and therefore increases the number of reactions it can catalyze.
The movement of Arg A145 and Phe A146 results in weaker ligand binding, and this form is known as the “coil” conformation (2). In contrast, the wild type or “helical” conformation is associated with tight ligand binding (2). Ligands that cause penicillin G acylase to adopt the “coil” conformation contain acid groups that become situated further away from the binding pocket, while ligands that bind the “helical” conformation are able to keep their acid groups entirely within the binding pocket (1). In the former group, the positioning of the acid groups allows for fewer hydrogen bonds to be formed (1). Thiopheneacetic acid is a ligand that complexes with penicillin G acylase in its “helical” conformation (1).It forms hydrogen bonds with the residues Asn B241, Ser B1, and Ala B69. This complex has been used to determine the crystal structure of penicillin G acylase. The binding pocket of penicillin G acylase contains many hydrophobic residues, which contributes to its preference for hydrophobic groups as substrates. Specifically, the aromatic groups of the binding pocket contribute highly to substrate recognition. The structure of this binding pocket is further defined by the binding of a calcium ion ligand (2). This ion bridges two Phe residues at A146 and B71, and is positioned at the lip of the active site (2). The pocket is constituted by a deep depression that results from the packing of the A chain onto the surface of the B chain (3). Each chain contains both alpha helices and beta sheets, with the helices flanking the anti-parallel sheets (3). Because these two types of secondary structure are generally separated into their own domains, penicillin G acylase is said to have an alpha + beta protein structure (2).
Through mutation analyses, it has been shown that Asn B241 helps maintain active site geometry and productive substrate binding in penicillin G acylase (2). Mutations at this residue inactivate the enzyme, as they are unable to stabilize the tetrahedral intermediate that forms during catalysis (2). This particular residue contributes to stability specifically by balancing the negative charge on the tetrahedral reaction intermediate (2). It also hydrogen bonds with penicillin G, and this important interaction is lost when there is a mutation at this site (2). Other residues that help to stabilize the intermediate are Gln B23 and Ala B69 (2).
The catalytic site of penicillin G acylase is located at Ser B1, and it functions via nucleophilic attack on the scissile amide (2). The nucleophilic attack is potentially mediated by a bridging water molecule (2). This process initiates the reaction mechanism for hydrolysis of penicillin G. The presence of the nucleophilic catalytic site at the N-terminus of the protein places penicillin G acylase in the N-terminal nucleophile hydrolase protein superfamily (2). This active site is covered by the linker peptides prior to its removal via cleavage (3).
The A chain of penicillin G acylase has a secondary structure that is 61% helical and 5% beta sheet. It contains 12 helices (consisting of 128 residues) and 3 strands of beta sheet (consisting of 12 residues). The B chain of penicillin G acylase has a secondary structure that is 25% helical and 27% beta sheet. It contains 18 helices (consisting of 141 residues) and 42 strands of beta sheet (consisting of 153 residues). There are also random coils present in penicillin G acylase.
Acyl-homoserine lactone acylase (PDB ID: 2WYD) contains similarities to penicillin G acylase. This protein comes from Pseudomonas aeruginosa, which is a bacterium like E. coli. Also like penicillin G acylase, acyl-homoserine lactone acylase is an N-terminal nucleophilic hydrolase with its active site in the fold of the A and B subunits of the heterodimer (7). It also has a hydrophobic binding pocket, but it is larger than that of penicillin G acylase to accommodate fatty acid chains (7). When the sequence of acyl-homoserine lactone acylase is compared with that of penicillin G acylase using BLAST, an E value of 3x10-8 is obtained. This value is significant because it is very small, and E values close to zero represent high sequence similarity, with a cutoff point for significance of .05. Additionally, BLAST gives a percent identity of 28%, meaning this percentage of residues match exactly between the two proteins. A Dali search gives a Z score of 15.7 and a root mean square deviation (RMSD) of 3.8 (8). The Dali search is used to find similarities in tertiary structure by comparing intramolecular distances. The RMSD reveals the divergence of two aligned structures from one another. This Z score is significant because it is greater than 2. These values confirm that these two proteins share similarities in both primary and tertiary structure. One difference between the two can be found in their ligands, as acyl-homoserine lactone acylase complexes with lauric acid and glycerol. Additionally, its A chain contains 170 amino acid residues and its B chain contains 546 residues. These two proteins have similar secondary structures. Like penicillin G acylase, the A chain of acyl-homoserine lactone acylase has a large helical portion and a small beta sheet portion (58% and 8%, respectively), and its B chain has moderate levels of both of these secondary structure types (25% and 31%, respectively).
Another similar protein is cephalosporin acylase (PDB ID: 1gk1), which is found in Pseudomonas sp. (another bacterium). This protein performs the same catalytic mechanism as penicillin G acylase, with the small difference of the presence of two catalytic dyads that do not occur in penicillin G acylase (His B23/Glu B455 and His B23/Ser B1) (9). The ligand with which cephalosporin acylase complexes is glycerol, like acyl-homoserine lactone acylase. A comparison of penicillin G acylase and cephalosporin acylase using BLAST yields an E value of 1x10-16, and Dali gives a Z score of 15 and an RMSD of 2.9 (8). This E value is, once again, close to zero, and so it represents a high level of sequence similarity. The Z score is well over 2, and so it is significant as well. A BLAST comparison also gives a percent identity of 22%, meaning this percentage of residues are identical between the two proteins. These values show that these two proteins contain similar elements in their primary and tertiary structures. Like acyl-homoserine lactone acylase, each chain of cephalosporin acylase is somewhat shorter than those of penicillin G acylase, as its A chain contains 153 amino acid residues and its B chain contains 522 residues. A key difference here is that cephalosporin acylase is actually a tetramer, not a dimer, as each of its A and B chains appear twice in the protein. Once again, there is similarity in secondary structure among the two proteins, as the A chain of cephalosporin acylase is largely helical and contains a small amount of beta sheet (64% and 5%, respectively), and its B chain has moderate levels of both of these secondary structure types (24% and 29%, respectively).