title: Inversion of the stereochemical configuration (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid: A computationally-assisted approach based on experimental evidence.
abstract: Clavulanic acid (CA), a potent inhibitor of β-lactamase enzymes, is produced by Streptomyces clavuligerus (Sc) cultivation processes, for which low yields are commonly obtained. Improved knowledge of the clavam biosynthetic pathway, especially the steps involved in the inversion of 3S-5S into 3R-5R stereochemical configuration, would help to eventually identify bottlenecks in the pathway. In this work, we studied the role of acetate in CA biosynthesis by a combined continuous culture and computational simulation approach. From this we derived a new model for the synthesis of N-acetyl-glycyl-clavaminic acid (NAG-clavam) by Sc. Acetylated compounds, such as NAG-clavam and N-acetyl-clavaminic acid, have been reported in the clavam pathway. Although the acetyl group is present in the β-lactam intermediate NAG-clavam, it is unknown how this group is incorporated. Hence, under the consideration of the experimentally proven accumulation of acetate during CA biosynthesis, and the fact that an acetyl group is present in the NAG-clavam structure, a computational evaluation of the tentative formation of NAG-clavam was performed for the purpose of providing further understanding. The proposed reaction mechanism consists of two steps: first, acetate reacts with ATP to produce a reactive acylphosphate intermediate; second, a direct nucleophilic attack of the terminal amino group of N-glycyl-clavaminic on the carbonyl carbon of the acylphosphate intermediate leads to a tetrahydral intermediate, which collapses and produces ADP and N-acetyl-glycyl-clavaminic acid. The calculations suggest that for the proposed reaction mechanism, the reaction proceeds until completion of the first step, without the direct action of an enzyme, where acetate and ATP are involved. For this step, the computed activation energy was ≅2.82kcal/mol while the reaction energy was ≅2.38kcal/mol. As this is an endothermic chemical process with a relatively small activation energy, the reaction rate should be considerably high. The calculations offered in this work should not be considered as a definite characterization of the potential energy surface for the reaction between acetate and ATP, but rather as a first approximation that provides valuable insight about the reaction mechanism. Finally, a complete route for the inversion of the stereochemical configuration from (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid is proposed, including a novel alternative for the double epimerization using proline racemase and NAG-clavam formation.
authors.affiliation: Grupo de Bioprocesos, Departamento de Ingeniería Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Howard
authors.initials: H
authors.lastname: Ramirez-Malule
authors.affiliation: Grupo de Química Física Teórica, Instituto de Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Albeiro
authors.initials: A
authors.lastname: Restrepo
authors.affiliation: Grupo de Química de Plantas Colombianas, Instituto de Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Wilson
authors.initials: W
authors.lastname: Cardona
authors.affiliation: Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK 24, 13355 Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Junne
authors.affiliation: Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK 24, 13355 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
authors.affiliation: Grupo de Bioprocesos, Departamento de Ingeniería Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia. Electronic address: rigoberto.rios@udea.edu.co.
authors.forename: Rigoberto
authors.initials: R
authors.lastname: Rios-Estepa
mesh_terms: D001372:Aza Compounds / Q000737:chemistry*; D008956:Models, Chemical; D015394:Molecular Structure; D013237:Stereoisomerism
chemical_list: D001372:Aza Compounds; C066045:proclavaminic acid
keywords: Accumulation; Acetate; Clavulanic acid; N-acetyl-glycyl-clavaminic acid; Potential energy surface; Reaction mechanism