Scouting lactamases for chemically stable lactams

The lactam motif a cyclic chemical structure embedding an essential amide bond is abundantly present in nature and occurs in various ring sizes. The smallest four-membered beta-lactams are notorious antibiotic drugs, which have received broad publicity in the past decades owing to widespread bacterial resistance against this class of antibiotics, causing a major clinical concern in the treatment of bacterial infections. Chemically, these molecules are relatively labile, leading to facile hydrolysis by specific enzymes, the beta-lactamases. The intermediate ring size of gamma- and delta-lactams results in contrast in a less strained structure, going in hand with resistance toward hydrolysis. As a consequence, their ring cleavage, which delivers important chemical building blocks in form of non-natural amino acids, necessitates harsh conditions (strong acid in concentrated boiling solutions) that eventually reduce the opportunities to utilize these motifs in synthesis. Clearly, milder alternatives applicable to synthetic routes are needed, in line with growing environmental concern and awareness for sustainable technologies. In the frame of a preliminary study, we could demonstrate the existence, in two bacterial strains, of catalytic machineries able to cleave such resistant cyclic structures. In this novelty-driven project, we aim to identify the enzymes responsible for the observed activity. In addition, under-explored enzymes active in the ring opening of lactam-like compounds will be studied. The project will focus on the following fundamental question: what structural/molecular requirements are necessary for these biological tools to be active on gamma- and delta-lactams? Our interdisciplinary approach will combine standard techniques and methodologies from chemistry, molecular biology, enzymology, genomics and biocatalysis. Through the identification of likely novel enzymes and investigation of rarely studied proteins, we wish to contribute to a better understanding of biological processes involved in the cleavage of lactam rings. We thereby aim at stimulating chemists to design novel synthetic protocols able to compete with nature in a more environmentally friendly approach.

The lactam motif - a cyclic chemical structure embedding an essential amide bond - is abundantly present in nature and occurs in various ring sizes. The smallest four-membered beta-lactams are notorious antibiotic drugs, which have received broad publicity in the past decades owing to widespread bacterial resistance against this class of antibiotics, causing a major clinical concern in the treatment of bacterial infections. Chemically, these molecules are relatively labile, leading to facile hydrolysis by specific enzymes, the beta-lactamases. The intermediate ring size of gamma- and delta-lactams results in contrast in a less strained structure, going in hand with resistance toward hydrolysis. As a consequence, their ring cleavage, which delivers important chemical building blocks in form of non-natural amino acids, necessitates harsh conditions (strong acid in concentrated boiling solutions). Clearly, milder alternatives are needed, in line with growing environmental concern and awareness for sustainable technologies. We tackled three different approaches in this project. i) We could exploit the catalytic power of bacterial strains to cleave such resistant cyclic structures. By feeding bacteria on the lactams, the cells developed the ability to metabolize the lactams. Several strains were successfully identified and subsequently utilized for the cleavage of several lactams. The genomes of these bacteria were sequenced and we started two approaches to identify the proteins responsible for the activity. This will be continued in a separate project. ii) Two enzymes were identified that work in tandem for the hydrolysis of lactams. They utilize a high energy compound called ATP, in combination with a simple molecule, bicarbonate, and water. This mechanism is complex and allows activation of the lactam, to facilitate the reaction. We could successfully employ this system on a variety of molecules. The reaction operates under mild conditions. iii) An enzyme rarely studied in the context of biocatalysis, and that is known to perform bond cleavage through activation of oxygen, was explored. We could demonstrate that while lactams are not reactive with this enzyme, it accepts some decorations on the natural substrate molecule. This protocol is an alternative to classical hydrolysis, in which water normally is used for cleaving bonds. To conclude, we could partially answer the question first raised at the on-set of the project: what structural/molecular requirements are necessary for biological tools to be active on medium-sized lactams? We have successfully identified enzymes involved in the cleavage of chemically stable lactam rings and shed some lights on the important aspects of these mechanisms, such as the need for external 'helper' molecules.