title: Structural insights into the synthesis of FMN in prokaryotic organisms.
abstract: Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyltransferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Instituto de Biocomputación y Física de Sistemas Complejos (Joint Unit BIFI-IQFR), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Herguedas
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Instituto de Biocomputación y Física de Sistemas Complejos (Joint Unit BIFI-IQFR), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: Isaias
authors.initials: I
authors.lastname: Lans
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Instituto de Biocomputación y Física de Sistemas Complejos (Joint Unit BIFI-IQFR), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Sebastián
authors.affiliation: GCMBE - Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Hermoso
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Instituto de Biocomputación y Física de Sistemas Complejos (Joint Unit BIFI-IQFR), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Martínez-Júlvez
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Instituto de Biocomputación y Física de Sistemas Complejos (Joint Unit BIFI-IQFR), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003352:Corynebacterium / Q000737:chemistry* / Q000201:enzymology; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008024:Ligands; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D005486:Flavin Mononucleotide; D017853:Phosphotransferases (Alcohol Group Acceptor); C022532:riboflavin kinase; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase
keywords: ATP:riboflavin kinase; FAD synthetase; conformational changes; crystal structure; molecular-dynamics simulations; substrate binding