Reconstitution of a split C-methylating module from the gladiolin trans-AT polyketide synthase Helen Smith 1,2 , Lona M. Alkhalaf 1 , Jozef R. Lewandowski 1 , Gregory L. Challis 1,3,4 , Matthew Jenner 1,3 1 Department of Chemistry, University of Warwick, UK, 2 Warwick Medical School, University of Warwick, UK, 3 Warwick Integrative Synthetic Biology Centre, University of Warwick, UK, 4 Monash University, Australia Trans -acyltransferase ( trans -AT) modular polyketide synthases (PKSs) are responsible for the biosynthesis of a structurally diverse range of polyketide natural products. The subunit interfaces in cis -AT PKS assembly lines are typically intermodular. 1 However, a particularly interesting feature of trans -AT PKSs is the prevalence of PKS subunit interfaces within modules, resulting in ‘split modules’. Recent work on subunit interfaces in trans- AT PKSs has led to some fascinating insights into ordered self-assembly of these megasynthases. 2-4 In this study, a previously uncharacterised subunit interface between a ketoreductase and a methyltransferase domain (KR/MT junction) from the gladiolin trans -AT PKS is examined. Whilst other PKS subunit interfaces have docking domains (DDs) at their C - and N -termini, which interact to facilitate communication and substrate transfer across the interface, bioinformatics analysis of the KR and MT domains from these split modules could not identify any potential DDs. The activity of module 7 from the gladiolin PKS (containing the KR/MT interface) has been investigated in vitro using a combination of mass spectrometry-based assays and mechanism-based covalent crosslinking. Chemically synthesised substrate mimics in combination with intact protein mass spectrometry have been used to characterise the enzymatic activity of the catalytic domains within the module and to probe the stereochemical course of the methylation and ketoreduction reactions. Carbene footprinting mass spectrometry experiments have identified specific regions involved in interaction across this interface.This information allowed crosstalk experiments to probe interaction between proteins at KR/MT interfaces from different trans -AT PKS pathways. KR/MT interfaces are prevalent in trans -AT PKSs, therefore insights from this work have the potential to guide future biosynthetic engineering efforts to help increase our understanding of why split modules are incorporated into such assembly lines.
Figure: Partial biosynthetic scheme for the production of gladiolin. Module 7 of thegladiolin PKS containing the KR/MT interface is shown on the left and the chemical transformations across this boundary are highlighted in blue on both the biosynthetic intermediate and the structure of gladiolin. References
1. Nat. Prod. Rep. 33, 231–316 (2016), 2. Nat. Chem. Biol. 14, 270–275 (2018), 3. ACS Chem. Biol. 11, 2466– 2474 (2016), 4. J. Am. Chem. Soc. 138, 4155–4167 (2016)
P74F
© The Author(s), 2022
Made with FlippingBook Learn more on our blog