Cell Biochemistry and Biophysics

, Volume 65, Issue 1, pp 57–68

Key Residues at the Riboflavin Kinase Catalytic Site of the Bifunctional Riboflavin Kinase/FMN Adenylyltransferase From Corynebacterium ammoniagenes

  • Ana Serrano
  • Susana Frago
  • Beatriz Herguedas
  • Marta Martínez-Júlvez
  • Adrián Velázquez-Campoy
  • Milagros Medina
Original Paper

DOI: 10.1007/s12013-012-9403-9

Cite this article as:
Serrano, A., Frago, S., Herguedas, B. et al. Cell Biochem Biophys (2013) 65: 57. doi:10.1007/s12013-012-9403-9

Abstract

Many known prokaryotic organisms depend on a single bifunctional enzyme, encoded by the RibC of RibF gene and named FAD synthetase (FADS), to convert Riboflavin (RF), first into FMN and then into FAD. The reaction occurs through the sequential action of two activities present on a single polypeptide chain where the N-terminus is responsible for the ATP:FMN adenylyltransferase (FMNAT) activity and the C-terminus for the ATP: riboflavin kinase (RFK) activity. Sequence and structural analysis suggest that T208, N210 and E268 at the C-terminus RFK module of Corynebacterium ammoniagenes FADS (CaFADS) might be key during RF phosphorylation. The effect of site-directed mutagenesis on the RFK activity, as well as on substrates and products binding, indicates that T208 and N210 provide the RFK active-site geometry for binding and catalysis, while E268 might be involved in the catalytic step as catalytic base. These data additionally suggest concerted conformational changes at the RFK module of CaFADS during its activity. Mutations at the RFK site also modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD. This observation supports the hypothesis that the hexameric assembly previously revealed by the crystal structure of CaFADS might play a functional role during catalysis.

Keywords

FAD synthetaseATP:riboflavin kinaseATP:FMN adenylyltransferaseSite-directed mutagenesisSubstrate bindingCatalytic activity

Abbreviations

FADS

FAD synthetase

RF

Riboflavin

FMN

Flavin mononucleotide

FAD

Flavin adenine dinucleotide

ATP

Adenosine 5′-triphosphate

RFK

ATP:riboflavin kinase

FMNAT

ATP:FMN adenylyltransferase

PIPES

1,4-Piperazine diethane sulphonic acid

ITC

Isothermal titration calorimetry

HPLC

High-performance liquid chromatography

UV

Ultra-violet

CD

Circular dichroism

WT

Wild-type

KFAD, KFMN

FAD and FMN fluorescence constants

Supplementary material

12013_2012_9403_MOESM1_ESM.pdf (686 kb)
Additional information includes; oligonucleotides for site-directed mutagenesis and methods for determination of kinetic and binding parameters; Table SD.1 with crystallographic data, Tables SD.2, SD.3 and SD.4 with thermodynamic parameters; Fig. SD.1 with scheme of the RFK and FMNAT activities of CaFADS; Fig. SD.2 with overall folding, topology and logo of sequence at the RFK consensus sequences; Fig. SD.3 with difference spectra; Fig. SD.4 with structural comparison of RFK modules; Fig. SD.5 with the trimeric structure of CaFADS. This material is available free of charge via the Internet at (pdf 686 kb)

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ana Serrano
    • 1
    • 2
  • Susana Frago
    • 1
    • 2
  • Beatriz Herguedas
    • 1
    • 2
  • Marta Martínez-Júlvez
    • 1
    • 2
  • Adrián Velázquez-Campoy
    • 1
    • 2
    • 3
  • Milagros Medina
    • 1
    • 2
  1. 1.Departamento de Bioquímica y Biología Molecular y Celular, Facultad de CienciasUniversidad de ZaragozaSaragossaSpain
  2. 2.Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit BIFI-IQFR (CSIC)Universidad de ZaragozaSaragossaSpain
  3. 3.Fundación ARAIDDiputación General de AragónSaragossaSpain