Biosynthesis of Beta Lactam Antibiotics

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Project Summary/Abstract The biosynthesis of three of the four “non-classical” clans of the b-lactam antibiotics will be investigated. Together with the fifth, or “classical” penicillins and cephalosporins, these drugs constitute >60% of the world antibiotic market and account for >$25B/yr in economic value. They remain vital mainstays of human health and longevity, but with their widespread use has come the inevitable rise of antibiotic-resistant infections. Structural modifications have slowed these effects, but there is increased reliance on the newer, non-classical families; for example, the b-lactamase inhibitor clavulanic acid and the potent, broad-spectrum carbapenems like Imipenem® and Meropenem,® inspired by the natural product thienamycin. The b-lactams are instructive examples of convergent evolution where the pathways to the five known classes exemplify remarkably different biosynthetic strategies, evolution of enzyme function to new tasks and impressive synthetic efficiency. In each of the three pathways to be investigated, remarkable, often unprecedented reactions, take place that will be studied using tools ranging from organic synthesis to enzymology, protein X-ray crystallography and in silico modeling to understand their enzyme mechanisms. This knowledge will be used to explore their potential for functional reprogramming and targeted mutation for the chemo-enzymatic synthesis of antibiotic variants of practical value. Much is known about structure/activity relationships among these drugs where manufacturing costs might be reduced by fermentation technology based on engineered biosynthetic enzymes and semi-synthesis. The enzymes of interest range from three cobalamin-dependent radical S-adenosylmethionine enzymes that we know now lie at the heart of carbapenem biosynthesis, to evolved domains of larger non-ribosomal peptide synthetases (NRPSs) that create b-lactam rings from peptide precursors in two strikingly distinct ways. One leads from a peptide seryl residue to the internal 4-membered ring of monocyclic b-lactam antibiotics while the other gives monobactams directly with their distinctive N- sulfonated b-lactam rings fully fledged. Renewed clinical interest attaches to this structural class for its clinically important property of resistance to Class B, or Zn++ metallo-b-lactamases, which can overmatch even the most potent carbapenems. We have recently shown the synthesis of the monobactam core is carried out in the C-terminal thioesterase (TE) domain of a NRPS. We have a crystal structure of this domain with a substrate mimic bound. Proposed are experiments to remodel the active site to accommodate stereoisomers of the native substrate to synthesize differently substituted monobactams. A combination of biochemical experiments, chemical crosslinking and x-ray crystallography will guide the engineering of a small library of TE domains for possible immobilization and the application of flow technology for larger scale synthesis.