title: 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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Andria L
authors.initials: AL
authors.lastname: Deaguero
authors.forename: Janna K
authors.initials: JK
authors.lastname: Blum
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Bommarius
mesh_terms: D019943:Amino Acid Substitution; D062365:Aminobenzoates / Q000378:metabolism; D000667:Ampicillin / Q000138:chemical synthesis*; D020134:Catalytic Domain; D002863:Chromogenic Compounds / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009579:Nitrobenzoates / Q000378:metabolism; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D062365:Aminobenzoates; D002863:Chromogenic Compounds; D029968:Escherichia coli Proteins; D009579:Nitrobenzoates; C008641:6-nitro-3-phenylacetamidobenzoic acid; D000667:Ampicillin; D010399:Penicillin Amidase; D019998:Racemases and Epimerases