title: Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm.
abstract: We recently reported a flavin-trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal-dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal-dependent Ftp catalysis in either Nqr- or Rnf-redox-containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD-binding protein to a Mg(2+)-dependent FAD pyrophosphatase (Ftp_Tp-like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated.
authors.affiliation: Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas, Texas, 75390.
authors.forename: Ranjit K
authors.initials: RK
authors.lastname: Deka
authors.affiliation: Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, Texas, 75390.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Brautigam
authors.affiliation: Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas, Texas, 75390.
authors.forename: Wei Z
authors.initials: WZ
authors.lastname: Liu
authors.affiliation: Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, Texas, 75390.
authors.forename: Diana R
authors.initials: DR
authors.lastname: Tomchick
authors.affiliation: Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas, Texas, 75390.
authors.forename: Michael V
authors.initials: MV
authors.lastname: Norgard
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000096:biosynthesis / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D020592:Shewanella / Q000201:enzymology* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D005486:Flavin Mononucleotide; D011755:Pyrophosphatases; C023417:FAD pyrophosphatase
keywords: FAD pyrophosphatase; FMN transferase; flavoprotein; lipoprotein; posttranslational modification; redox protein