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

International Journal of Molecular Sciences

Milagros Medina¹  Ana Serrano¹  Sonia Arilla-Luna¹ 

[1] Department of Biochemistry and Molecular and Cellular Biology, Faculty of Sciences, and Institute of Biocomputation and Physics of Complex Systems (Joint Units: BIFI-IQFR and GBsC-CSIC), University of Zaragoza, 50009 Zaragoza, Spain;

关键词: prokaryotic fad synthase;    species-specific traits;    adenylyltransferase activity;    head-to-tail organization;    isothermal titration calorimetry;    site-directed mutagenesis;    biosynthesis;    steady-state kinetics;   

DOI  :  10.3390/ijms20205083

来源: DOAJ

【 摘 要 】

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.

【 授权许可】

Unknown