Catalytic mechanism and molecular engineering of quinolone biosynthesis in dioxygenase AsqJ

Sophie L Mader,Ville R I Kaila,Michael Groll,Alois Bräuer

doi:10.1038/s41467-018-03442-2

Sophie L Mader, Ville R I Kaila + Show 2 more

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https://doi.org/10.1038/s41467-018-03442-2

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Abstract

The recently discovered FeII/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans stereoselectively catalyzes a multistep synthesis of quinolone alkaloids, natural products with significant biomedical applications. To probe molecular mechanisms of this elusive catalytic process, we combine here multi-scale quantum and classical molecular simulations with X-ray crystallography, and in vitro biochemical activity studies. We discover that methylation of the substrate is essential for the activity of AsqJ, establishing molecular strain that fine-tunes π-stacking interactions within the active site. To rationally engineer AsqJ for modified substrates, we amplify dispersive interactions within the active site. We demonstrate that the engineered enzyme has a drastically enhanced catalytic activity for non-methylated surrogates, confirming our computational data and resolved high-resolution X-ray structures at 1.55 Å resolution. Our combined findings provide crucial mechanistic understanding of the function of AsqJ and showcase how combination of computational and experimental data enables to rationally engineer enzymes.

Highlights

The recently discovered FeII/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans stereoselectively catalyzes a multistep synthesis of quinolone alkaloids, natural products with significant biomedical applications
In order to probe the energetics and structure of the reaction catalyzed by AsqJ for its natural substrate 1, we performed quantum chemical density functional theory (DFT) calculations on active site enzyme models of AsqJ (Fig. 1a, inset)
The catalytic cycle of AsqJ is initiated by binding of dioxygen to the FeII active site

Summary

Introduction

The recently discovered FeII/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans stereoselectively catalyzes a multistep synthesis of quinolone alkaloids, natural products with significant biomedical applications. We demonstrate that the engineered enzyme has a drastically enhanced catalytic activity for non-methylated surrogates, confirming our computational data and resolved high-resolution X-ray structures at 1.55 Å resolution. The non-heme FeII/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans (Fig. 1a) is an exceptional enzyme that activates dioxygen and stereoselectively catalyzes a C–C bond desaturation and epoxidation reaction[1,2]. In addition to the central role of non-heme FeII/α-ketoglutarate-dependent oxygenases in many metabolic pathways[7,8,9], elucidation of the catalytic mechanism of AsqJ is important due to the promising application of different quinol alkaloids as potential drug candidates. From a chemical point of view this finding is unexpected, since the methyl group is located three bonds apart from the reacting atoms in the substrate

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