The Biosynthesis of Flavin Cofactors in Listeria monocytogenes

Abstract

Listeria 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.

Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes

“…Thus, the internalization of the isoalloxazine ring and the formation of the competent complex for catalysis at the FMANT site of Ca FADS are highly sensitive to subtle changes in the conformation of the isoalloxazine binding cavity, indicating that there is fine regulation of the binding of the flavin substrate for efficient FMNAT catalysis. Such an idea is in line with former studies showing that some FADSs are only able to internalize and, as a consequence, to transform flavins at their FMNAT site when they are in their reduced state [17], while in others, the transformation of both oxidized or reduced flavins occurs with different catalytic efficiencies [16].…”

“…Nonetheless, recent studies using other family members, such as the FADSs from Streptococcus pneumoniae ( Sp FADS) and Listeria monocytogenes ( Lm FADS), envisage species-specific traits that modulate their efficiency. The reported differences among prokaryotic FADSs include (i) the stabilization or not of quaternary organizations during catalysis, (ii) the differential modulation of the RFK activity by substrates and products, (iii) the influence of the redox environment in the occurrence and/or efficiency of the RFK and/or FMNAT activities, and (iv) cooperation in the binding of substrates and products [14,16,17,18,19,20]. These data exemplify alternative strategies for FMN and FAD biosynthesis and homeostasis in different bacteria and, in some of them, they point to the FMNAT activity as a “redox sensor” of the media.…”

“…Together with site-directed mutational studies on conserved motifs in both the FADS and NT families, these models have pointed to several residues on the FMNAT module of Ca FADS as responsible for the stabilization of ATP and for the transfer of its nucleotidyl group to FMN [12]. In addition, variations in residues both coating the flavinic binding site and the channel between the ATP:Mg 2+ and FMN binding sites at the FMNAT module have been claimed as responsible for the variability in requirements for FAD biosynthesis among prokaryotic proteins from different species [16,17].…”

Human riboflavin kinase: Species‐specific traits in the biosynthesis of the FMN cofactor

“…This later behavior resulted similar to that described for CaFADS and SpnFADS, but differed for other species, as Listeria monocytogenes, whose turnover and efficiency highly depended on the redox status of the media. 19,22,23,26,27 Steady-state rates for the HsRFK activity showed saturation profiles for both substrates (Figure 2A (Table 1), respectively. Furthermore, no inhibition by excess of RF substrate was detected, contrary to that reported for the RFK activity of CaFADS 31 but in line with previous data for HsRFK, 42 SpnFADS, 19 and LmFADS-1.…”

“…FMN is a more potent inhibitor in CaFADS (K RF M /K FMN I = 7.1) than in SpnFADS or HsRFK (0.8 and 1, respectively); and also, RF is a very strong inhibitor of the RFK activity in CaFADS but not in the other enzymes. 19,20,27 Therefore, FMN biosynthesis in Homo sapiens will be regulated by the products of the RFK reaction. In addition, and considering that transport across membranes appears favored for the RF substrate over FMN and FAD, intracellular compartmentalization with different substrate/ product concentrations and/or the presence of different isoforms might also apply in the in vivo HsRFK regulation.…”