July 29, 2019 -- Determining how antibiotic structures are formed in nature is crucial knowledge that scientists will leverage in the development of the next generation of antibiotics. Researchers from Washington University in St. Louis and the University at Buffalo published the X-ray crystal structure of an enzyme that produces a candidate antibiotic in Nature Communications on July 31, 2019.
Obafluorin (ObiF) is an N-acyl-α-amino-β-lactone with broad spectrum antimicrobial activity. It was discovered in 1984 by the Squibb Institute, is made by a fluorescent strain of soil bacteria, Pseudomonas fluorescens, that forms biofilms on plant roots. Instead of being assembled by a ribosome, biosynthesis of the ObiF peptide occurs by non-ribosomal peptide synthetase (NRPS), which only synthesizes one type of peptide. NRPS act on demand as secondary metabolites are needed for cellular function. β-lactone within ObiF is produced from dihydroxybenzoic acid and a β-hydroxy amino acid that cyclizes into the β-lactone during product release of the NRPS. β-lactone is a key formation that allows for turnover of the catalytic cycle of ObiF.
About “Enchanted Rings”
Penicillin and related antibiotics contain an “enchanted ring,” called the β-lactam ring. Antibiotics that include these rings are arguably the most important drugs in human history; increasing global life expectancy by an estimated five years. The antimicrobial activity of these antibiotics is derived from the unstable four-membered rings contained in their structures. Similar to penicillin, ObiF contains a four-membered ring, β-lactone, which inhibits a large class of enzymes called the serine hydrolases. According to Timothy Wencewicz, the principle investigator of this research, “the strain turns these rings into molecular bombs that go off when they are put in the right place at the right time, which is useful for killing microbes.”
Determine Structure and Bioactive Components of NRPS
Scientists set out to validate the importance of the nonribosomal peptide synthetase (ObiF1) structure and how the configuration is used to enable β-lactone cyclization within its thioesterase (TE) domain. Using biochemical assays to determine key steps in the catalytic cycle and x-ray crystallography to determine structure, they ascertained the structure of ObiF1 with the β-lactone-producing TE domain and an interaction between the C-terminal MbtH-like domain with an upstream adenylation domain. There are conserved residues in these regions including a rare catalytic amino acid that generates a reactive thioester intermediate. This unusual chemistry allows the NRPS to overcome the energy barrier that otherwise prevents the formation of a strained ring.
X-ray crystallography: a technique used for determining atomic and molecular structure of a crystal, where the structure causes a beam of X-rays to diffract in specific directions. By measuring diffraction angles and intensities the three-dimensional density of electrons and positions of atoms can be determined. This procedure required crystallization which requires a purified sample, followed by exposure to an intense beam of X-rays, and finally by computational analysis of chemical information to determine arrangement of atoms.
Reconstruct Natural Processes in the Laboratory
Effectively, the researchers used genetics to determine the biosynthetic machinery that bacteria use make ObiF and therefore how to synthesize it in the laboratory. They seek to quickly and easily create analogs of the natural product in the laboratory to optimize its molecular properties and bioactivity. The ultimate goal is to achieve synthetic production of catalytic peptides containing β-lactones, such as ObiF, as potential new antibiotic therapeutics to be used in drug discovery.
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