Improving the diastereoselectivity of penicillin G acylase for ampicillin synthesis from racemic substrates

Abstract

Semi-synthetic β-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the β24 position. βPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, βPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. βPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

Structure mediation in substrate binding and post‐translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase

“…Mutational studies of residues α146F, β24F, and β71F have already been reported in Ec PGA . Various complexes of phenylacetic acid and (R)‐α‐methylphenylacetic acid with wild‐type and mutants of Ec PGA have confirmed the role of α146F and β24F in substrate selectivity and diastereoselectivity . Similarly, it was demonstrated that replacing β71F selectively with other amino acids modified the catalytic properties and enantioselectivity of Ec PGA.…”

“…(C,D)]. Andrew et al . have previously reported the crucial role residues Phe146 and Phe24 play in diastereoselectivity.…”

Protein Engineering of d‐Succinylase from Cupriavidus sp. for d‐Amino Acid Synthesis and the Structural Implications

“…At first, we selected a target region to be mutated based on the available literature [11,12,17–20] . Doggleby et al.…”

“…and Morillas et al. had also described the function of the mutant, Penicillin acylase F24β from Esherichia coli and Penicillin acylase F71β from E. coli with improved diastereoselectivity and substrate specificity, respectively [17,18] . In addition, Tishkov et al.…”

Computational Design of a pH Stable Enzyme: Understanding Molecular Mechanism of Penicillin Acylase's Adaptation to Alkaline Conditions

“…These enzymes are members of the N-terminal nucleophile (Ntn) hydrolase superfamily, which is characterized by a common catalytic N-terminal nucleophile [20]. They are also capable of effective and enantioselective acylation of amino compounds in aqueous medium and can be used for preparation of individual enantiomers of primary amines, amino alcohols, non-conventional amino acids and aminonitriles [21]–[28].…”