60970 20190709135501.0 doi 10.1038/srep43880 sideral 98517 ART-2017-98517 eng Huijbers, M. M. E. Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor 2017 Access copy available to the general public Unrestricted Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ¿ABC, which lacks helices aA, aB and aC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered. info:eu-repo/grantAgreement/ES/MICINN/BIO2013-42978-P info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 4.122 2017 MULTIDISCIPLINARY SCIENCES 12 / 64 = 0.188 2017 Q1 T1 1.533 2017 Multidisciplinary 2017 Q1 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion (orcid)0000-0001-9047-0046 Martínez-Júlvez, M. Universidad de Zaragoza Westphal, A. H. Delgado-Arciniega, E. (orcid)0000-0001-8743-0182 Medina, M. Universidad de Zaragoza Van Berkel, W. J. H. 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 7 (2017), 43880 [13 pp.] Sci. rep. Scientific reports 2045-2322 1365790 http://zaguan.unizar.es/record/60970/files/texto_completo.pdf Versión publicada 110249 http://zaguan.unizar.es/record/60970/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:60970 articulos driver 2019-07-09-11:46:34 ARTICLE