Abstract
In bacteria, riboflavin phosphorylation and subsequent conversion of FMN into FAD are carried out by FAD synthetase, a single bifunctional enzyme. Both reactions require ATP and Mg(2+). The N-terminal domain of FAD synthetase appears to be responsible for the adenylyltransferase activity, whereas the C-terminal domain would be in charge of the kinase activity. Binding to Corynebacterium ammoniagenes FAD synthetase of its products and substrates, as well as of several analogues, is analyzed. Binding parameters for adenine nucleotides to each one of the two adenine nucleotide sites are reported. In addition, it is demonstrated for the first time that the enzyme presents two independent flavin sites, each one related with one of the enzymatic activities. The binding parameters of flavins to these sites are also provided. The presence of Mg(2+) and of both adenine nucleotides and flavins cooperatively modulates the interaction parameters for the other ligands. Our data also suggest that during its double catalytic cycle FAD synthetase must suffer conformational changes induced by adenine nucleotide-Mg(2+) or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains.
Highlights
The two catalytic cycles of FAD synthetase (FADS) involve the binding of two ATP, one RF, and one FMN molecules as substrates and the production of one ADP, one PPi, one FMN, and one FAD
This study provides a thermodynamic characterization of substrate/product binding to CaFADS and of its individually cloned C terminus domain, analogous in sequence and structure to the eukaryotic riboflavin kinase (RFK) [15]
Our data confirm that CaFADS holds two different adenine nucleotide-binding sites, each of them involved in each one of the enzyme catalytic activities [10]
Summary
The presence of Mg2ϩ improves the turnover of both processes but, low concentrations (Ͻ1 mM) enhance the kinase activity, much larger concentrations (ϳ10 mM) are required for maximal FAD production [8] These studies indicated the presence of two independent ATP-binding sites, one at the RF phosphorylation site and one at the FMN-adenylylation site, but a single pocket was proposed to allocate the isoalloxazine-ribityl moieties of both substrates, RF and FMN, in the two reactions [10]. The asymmetric unit is a dimer, it does not appear functional These observations, together with the fact that the FMN produced in the phosphorylation process has to be released before rebinding as substrate for the second reaction [10], make it logical to propose the presence of a second flavinbinding site, located in the N-terminal domain. The structural model of FADS from Corynebacterium ammoniagenes (CaFADS) shows the main structural arrangements present in TmFADS [13, 14], and it shows the disposition of some
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