Molecular insights into the mechanism of substrate binding and catalysis of bifunctional FAD synthetase from Staphylococcus aureus.

Anusree, Lohithakshan and Raja, Narayanasamy and Upasana, S. Potteth and Shwetha, Keshava and Vibha, Nagaraja and Usharani, D. and Ravi, Kumar (2021) Molecular insights into the mechanism of substrate binding and catalysis of bifunctional FAD synthetase from Staphylococcus aureus. Biochimie, 182. pp. 217-227. ISSN 0300-9084

PDF Biochimie 182 (2021) 217e227.pdf - Published Version Restricted to Registered users only Download (2MB) | Request a copy

Abstract

Flavin adenine dinucleotide synthetase (FADS), a bifunctional prokaryotic enzyme, is involved in the synthesis of two vital cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Here, we investigated the biochemical characteristics of FADS from Staphylococcus aureus (Sa), a pathogenic bacteria causing food-borne diseases. The SaFADS possesses riboflavin kinase (RFK) and FMN adenylyltransferase (FMNAT) activities that transforms riboflavin to FMN and FMN to FAD, respectively. The FMNAT domain also exhibits reversible FAD pyrophosphorylase activity (FADpp). Further, we show that the FMNAT and FADpp activities are dependent on the reducing environment. Mutations of the conserved K289 and F290 residues present on the RFK domain affect the kinetic parameters of both the RFK and FMNAT domains. Additionally, the molecular dynamics analysis of apo and riboflavin: ATP: Mg2þ ternary complex of SaFADS shows that F290 is involved in stabilizing the active site geometry to hold the enzyme-substrate complex. In addition, the deletion of the ah2 helix that acts as a connecting linker between the FMNAT and RFK domains showed substantial loss of their activities. The helix deletion could have affected the flap motion of L2c, L4c, b4n and L3n present in the close proximity resulting in the distortion of the active site geometry. In conclusion, our study has characterized the RFK and FMNAT activities of SaFADS and shown the importance of conserved K289 and F290 in RFK activity. As FADSs are potential drug targets, understanding their mechanism of action might help in discovering speciesspecific antibacterial drugs.

Item Type:	Article
Uncontrolled Keywords:	FAD synthetase Molecular dynamics FMNAT RFK domain
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 07 Enzyme Biochemistry
Divisions:	Food Safety Analytical Quality Control Lab Molecular Nutrition
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	21 Feb 2022 04:44
Last Modified:	21 Feb 2022 04:44
URI:	http://ir.cftri.res.in/id/eprint/15127

Actions (login required)

View Item