Molecular Choreography Of An Antibiotic Assembly Line

Abstract

Plants and microorganisms produce a vast array of structurally complex and diverse antibacterial, antiviral, antitumor and antimetabolite substances with wide‐ranging human and animal health applications. In fact the majority of compounds used clinically and agriculturally in the United States are either natural products or semi‐synthetic derivatives thereof. Polyketides are an important class of natural compounds that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. Their biosynthetic machinery, represented by type I polyketide synthases, has an architecture in which successive modules catalyze two‐carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We recently employed cryo‐electron microscopy (cryo‐EM) to visualize a full‐length module from the biosynthetic pathway that creates the antibiotic pikromycin. The cryo‐EM 3D reconstructions revealed a totally unexpected architecture compared to the homologous dimeric mammalian fatty acid synthase. The dimeric PKS module creates a single reaction chamber for the two acyl carrier protein (ACP) domains that carry building blocks and intermediates between acyltransferase (AT), ketosynthase (KS), and ketoreductase (KR) active sites. By trapping the PKS module in each of several biochemical states we obtained 3D maps that recapitulate its full enzymatic cycle. The ACP domains are differentially and precisely positioned after polyketide chain substrate loading on the active site of KS, after extension to the b‐keto‐intermediate, and after b‐hydroxy product generation. The structures reveal the ACP dynamics for sequential binding to catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. This study highlights the conformational transitions coupled to substrate transfer and modification in a way that facilitates high throughput unidirectional substrate processing, and establishes a new model for molecular dissection of these multifunctional enzyme systems.