Evolutionary divergence of chloroplast FAD synthetase proteins

<p>Abstract</p> <p>Background</p> <p>Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history.</p> <p>Results</p> <p>Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The <it>C</it>-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the <it>N</it>-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval <it>et al</it>. in 2008. Furthermore, our observations and preliminary experimental results indicate that the <it>C-</it>terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the <it>C</it>-terminus.</p> <p>Conclusions</p> <p>A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose <it>C</it>-terminal module might be involved in a distinct catalytic activity.</p

The biosynthesis of flavin cofactors in Listeria monocytogenes

55 Pags.- 7 Figs.- Suppl. Mat. (9 Figs.- 5 Tabls.). The definitive version is available at: https://www.sciencedirect.com/science/journal/00222836Listeria monocytogenes is riboflavin auxotrophic, but it has two genes envisaged to transform riboflavin into FMN and FAD after its uptaked by specialized transporters. One encodes a bifunctional type I FAD synthase (FADS, herein LmFADS-1), while the other produces a protein similar to type I at the FMN:ATP adenylyltransferase (FMNAT) site but with a shorter C-terminal that lacks any riboflavin kinase (RFK) motif. This second protein is rare among bacteria and has been named FADS type II (LmFADS-2). Here we present a biochemical and biophysical study of LmFADS-1 and LmFADS-2 by integrating kinetic and thermodynamic data together with sequence and structural prediction methods to evaluate their occurrence in Listeria, as well as their function and molecular properties. Despite LmFADS-1 similarities to other type I FADSs, (i) its RFK activity has not riboflavin substrate inhibition and occurs under reducing and oxidizing conditions, (ii) its FMNAT activity requires strong reducing environment, and (iii) binding of reaction products, but not substrates, favors binding of the second ligand. LmFADS-2 produces FAD under oxidizing and reducing environments, but its C-terminus module function remains unknown. Listeria species conserve both FADSs, being sequence identity high within L. monocytogenes strains. Our data exemplify alternative strategies for FMN and FAD biosynthesis and homeostasis, envisaging that in Listeria two FADSs might be required to fulfill the supply of flavin cofactors under niches that can go from saprophytism to virulence. As FADSs are attractive antimicrobial targets, understanding of FADSs traits in different species is essential to help in the discovery of specific antimicrobials.This work has been supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) (BIO2016-75183-P AEI/FEDER, UE to M.M.) and the Government of Aragón-FEDER (E35_17R to M.M.).Peer reviewe