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

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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.

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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

K FAD, K FMN :

FAD and FMN fluorescence constants

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Acknowledgments

This work has been supported by the Spanish Ministry of Science and Innovation [BIO2010-14983 to M.M.].

Author information

Authors and Affiliations

  1. Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna, 12, 50009, Saragossa, Spain

    Ana Serrano, Susana Frago, Beatriz Herguedas, Marta Martínez-Júlvez, Adrián Velázquez-Campoy & Milagros Medina

  2. Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Mariano Esquillor s/n, Campus Río Ebro, 50018, Saragossa, Spain

    Ana Serrano, Susana Frago, Beatriz Herguedas, Marta Martínez-Júlvez, Adrián Velázquez-Campoy & Milagros Medina

  3. Fundación ARAID, Diputación General de Aragón, Saragossa, Spain

    Adrián Velázquez-Campoy

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  1. Ana Serrano
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  2. Susana Frago
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  3. Beatriz Herguedas
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  6. Milagros Medina
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Corresponding author

Correspondence to Milagros Medina.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

12013_2012_9403_MOESM1_ESM.pdf

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)

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Serrano, A., Frago, S., Herguedas, B. et al. Key Residues at the Riboflavin Kinase Catalytic Site of the Bifunctional Riboflavin Kinase/FMN Adenylyltransferase From Corynebacterium ammoniagenes . Cell Biochem Biophys 65, 57–68 (2013). https://doi.org/10.1007/s12013-012-9403-9

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  • DOI: https://doi.org/10.1007/s12013-012-9403-9

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