Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase
Huerta, Carlos, Jr.
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Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme FAD and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different set of active site residues to accomplish the same chemistry. Extensive biochemical studies from endogenous bacterial and mammalian FMNATs using FMN analogs and various cations have suggested that the architectures of the substrate binding and catalytic sites are different. Sequence comparison reveals that eukaryotic FMNAT is related to the PAPS reductase-like family, which belongs to the adenine nucleotide α hydrolase-like superfamily. Despite the classification of eukaryotic FMNAT, the residues involved in substrate binding and catalysis are not completely known, as eukaryotic FMNAT has no sequence similarity to other known flavin binding proteins. To determine the unique flavin binding site, and to investigate the residues involved in substrate binding and the mechanism of catalysis, we utilized X-ray crystallography and biochemical methods. Here we report the first structural characterization of a eukaryotic FMNAT from a pathogenic yeast Candida glabrata (CgFMNAT). Four crystal structures of CgFMNAT in different complexed forms were determined at resolutions between 1.20-1.95 Å, capturing the enzyme active site states prior to and after catalysis. These structures reveal a novel flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNAT provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. The different complexed forms of CgFMNAT allowed a structure-based investigation into the kinetic properties of eukaryotic FMNAT, whereby two "supermutants" were identified from mutagenic analysis. The steady-state kinetics and product inhibition properties of the two "supermutants" provided a basis for understanding the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms.