The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

Ana Serrano,Silvia Baquedano,Adrián Velázquez-Campoy,María Sebastián,Beatriz Herguedas,Sonia Arilla-Luna,Milagros Medina,Marta Martínez-Júlvez

doi:10.1038/s41598-017-00402-6

Abstract

Bifunctional FAD synthetases (FADSs) fold in two independent modules; The C-terminal riboflavin kinase (RFK) catalyzes the RFK activity, while the N-terminal FMN-adenylyltransferase (FMNAT) exhibits the FMNAT activity. The search for macromolecular interfaces in the Corynebacterium ammoniagenes FADS (CaFADS) crystal structure predicts a dimer of trimers organization. Within each trimer, a head-to-tail arrangement causes the RFK and FMNAT catalytic sites of the two neighboring protomers to approach, in agreement with active site residues of one module influencing the activity at the other. We analyze the relevance of the CaFADS head-to-tail macromolecular interfaces to stabilization of assemblies, catalysis and ligand binding. With this aim, we evaluate the effect of point mutations in loop L1c-FlapI, loop L6c, and helix α1c of the RFK module (positions K202, E203, F206, D298, V300, E301 and L304), regions at the macromolecular interface between two protomers within the trimer. Although none of the studied residues is critical in the formation and dissociation of assemblies, residues at L1c-FlapI and helix α1c particularly modulate quaternary architecture, as well as ligand binding and kinetic parameters involved with RFK and FMNAT activities. These data support the influence of transient oligomeric structures on substrate accommodation and catalysis at both CaFADS active sites.

Highlights

Many biological events require the formation of protein oligomers or multiprotein assemblies, rendering this formation a mechanism for the modulation of protein activity[1,2,3]
Most mutants primarily purified as monomers, with a small population of oligomeric species (Figure SP1), similar to WT CaFADS8, 19
Some mutations considerably decreased the intensity of the near-UV circular dichroism (CD) signal (Figures SP2C and SP2D), suggesting local changes in the region contributing to the signal

Summary

Introduction

Many biological events require the formation of protein oligomers or multiprotein assemblies, rendering this formation a mechanism for the modulation of protein activity[1,2,3]. At the molecular level, we analyze the relevance of the trimer head-to-tail interfaces in the stabilization of the quaternary assembly, as well as in ligand binding and catalysis With this aim, we evaluate the effects produced by point mutations at L1c-FlapI (K202, E203 and F206), L6c (D298), and α1c (V300, E301 and L304) of CaFADS (Figure 1)[6]. We evaluate the effects produced by point mutations at L1c-FlapI (K202, E203 and F206), L6c (D298), and α1c (V300, E301 and L304) of CaFADS (Figure 1)[6] These secondary structural elements belong to the RFK module and contribute to the structure of the active sites for FMN and FAD synthesis of two contiguous protomers within each trimer (Figure 1A)[6]. Our results support the formation of organized transient oligomeric assemblies in the catalytic cycles of CaFADS, and indicate that they contribute to modulating the CaFADS efficiency during catalysis

Methods

Results

Discussion

Conclusion