Structure-Based Analysis of Transient Interactions between Ketosynthase-like Decarboxylase and Acyl Carrier Protein in a Loading Module of Modular Polyketide Synthase

Abstract

Ketosynthase-like decarboxylase (KS Q ) domains are widely distributed in the loading modules of modular type I polyketide synthases (PKSs) and catalyze the decarboxylation of the (alkyl-)malonyl unit bound to the acyl carrier protein (ACP) in the loading module for the construction of the PKS starter unit. Previously, we performed a structural and functional analysis of the GfsA KS Q domain involved in the biosynthesis of macrolide antibiotic FD-891. We furthermore revealed the recognition mechanism for the malonic acid thioester moiety of the malonyl-GfsA loading module ACP (ACP L ) as a substrate. However, the exact recognition mechanism for the GfsA ACP L moiety remains unclear. Here, we present a structural basis for the interactions between the GfsA KS Q domain and GfsA ACP L . We determined the crystal structure of the GfsA KS Qacyltransferase (AT) didomain in complex with ACP L (ACP L =KS Q AT complex) by using a pantetheine crosslinking probe. We identified the key amino acid residues involved in the KS Q domain−ACP L interactions and confirmed the importance of these residues by mutational analysis. The binding mode of ACP L to the GfsA KS Q domain is similar to that of ACP to the ketosynthase domain in modular type I PKSs. Furthermore, comparing the ACP L =KS Q AT complex structure with other full-length PKS module structures provides important insights into the overall architectures and conformational dynamics of the type I PKS modules.

How Acyl Carrier Proteins (ACPs) Direct Fatty Acid and Polyketide Biosynthesis

“…In contrast, the KS domain seems to be more specific towards the ACP domain, which has been shown by structural data of three different type I PKSs describing the elongation interface [66,67,98] . A similar binding mode was observed between the KS Q and ACP domain in a PKS loading module [99] …”

“…[66,67,98] A similar binding mode was observed between the KS Q and ACP domain in a PKS loading module. [99] It has been postulated in 2010 that the ACP docks at a distinct position of the KS for the translocation reaction, [100] but gaining direct structural understanding of the translocation step remains a challenge. To date, several biochemical studies addressed translocation, [70,[101][102][103][104][105] but only a preliminary structural estimation on the ACP-KS translocation complex has been provided for the arched structure of PikAIII at 8.6 Å.…”

Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

“…[66,67,98] Eine ähnliche Bindungsweise wurde zwischen der KS Q -und der ACP-Domäne in einem PKS-Lademodul beobachtet. [99] Im Jahre 2010 wurde postuliert, dass das ACP an einer bestimmten Position der KS während der Translokationsreaktion bindet, [100] aber das direkte strukturelle Verständnis des Translokationsschritts bleibt eine Herausforderung. Bis heute wurden mehrere biochemische Studien zur Translokation durchgeführt, [70,[101][102][103][104][105] aber nur eine vorläufige strukturelle Näherung des ACP-KS-Translokationskomplexes konnte für die gewölbte Struktur von PikAIII bei 8,6 Å bereitgestellt werden.…”

“…Im Gegensatz dazu scheint die KS‐Domäne spezifischer bezüglich der ACP‐Domäne zu sein, was durch strukturelle Daten von drei verschiedenen Typ I PKSs beschrieben wurde, die die Elongationsreaktion beschreiben [66,67,98] . Eine ähnliche Bindungsweise wurde zwischen der KS Q ‐ und der ACP‐Domäne in einem PKS‐Lademodul beobachtet [99] …”

The structure of full-length AFPK supports the ACP linker in a role that regulates iterative polyketide and fatty acid assembly

“…Characteriza[on of the structural framework suppor[ng polyke[de produc[on remains challenging 21,22 . Individual AT or KS-AT didomains have been characterized bound to ACP through crosslinking, with synthe[c covalent tethers, or as isolated apo-ACP complexes and reveal small charge complementary interfaces [23][24][25][26][27][28][29][30] . Rare full-length structures ini[ally failed to capture the ACP domain due to a flexible linker to the main mega-enzyme, the inherent transient nature of the ACP-pPant for each enzyme interface 31,32 .…”