Insights into the role of F26 residue in the FMN: ATP adenylyltransferase activity of Staphylococcus aureus FAD synthetase

Anusree, Lohithakshan and Raja, Narayanasamy and Prashant, Deshmukh and Dandamudi, Usharani and Ravi, Kumar (2022) Insights into the role of F26 residue in the FMN: ATP adenylyltransferase activity of Staphylococcus aureus FAD synthetase. BBA - Proteins and Proteomics, 1870. pp. 1-11.

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Abstract

The bifunctional flavin adenine dinucleotide synthetase (FADS) synthesizes the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) co-factors essential for the function of flavoproteins. The Staphylococcus aureus FADS (SaFADS) produces FMN from riboflavin (RF) by ATP:riboflavin kinase (RFK) activity at its C-terminal domain. The N-terminal domain converts FMN to FAD under a reducing environment by FMN:ATP adenylyltransferase (FMNAT) activity which is reversible (FAD pyrophosphorylase activity). Herein, we investigated the role of F26 residue of the 24-GFFD-28 motif of SaFADS FMNAT domain, mostly conserved in the reducing agent-dependent FADSs. The steady-state kinetics studies showed changes in the KmATP values for mutants, indicating that the F26 residue is crucial for the FMNAT activity. Further, the FMNAT activity of the F26S mutant was observed to be higher than that of the wild-type SaFADS and its other variants at lower reducing agent concentration. In addition, the FADpp activity was inhibited by an excess of FAD substrate, which was more potent in the mutants. The altered orientation of the F26 side-chain observed in the molecular dynamics analysis suggested its plausible involvement in stabilizing FMN and ATP substrates in their respective binding pockets. Also, the SaFADS ternary complex formed with reduced FMN exhibited significant structural changes in the β4n-β5n and L3n regions compared to the oxidised FMN bound and apo forms of SaFADS. Overall, our data suggests the functional role of F26 residue in the FMNAT domain of SaFADS.

Item Type:	Article
Uncontrolled Keywords:	FAD synthetase, Riboflavin kinase activity, FMN adenylyltransferase activity, Steady-state kinetics, Molecular dynamics
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 02 Bacteriology
Divisions:	Molecular Nutrition Food Safety Analytical Quality Control Lab
Depositing User:	Somashekar K S
Date Deposited:	06 Mar 2025 06:09
Last Modified:	06 Mar 2025 06:09
URI:	http://ir.cftri.res.in/id/eprint/19214

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