title: Structure and function of YcnD from Bacillus subtilis, a flavin-containing oxidoreductase.
abstract: YcnD from the gram-positive bacterium Bacillus subtilis is a member of a family of bacterial proteins that act as NADH- and/or NADPH-dependent oxidoreductases. Here, we report for the first time on the biochemical characterization of the purified protein, demonstrating that YcnD is an FMN-containing enzyme that can be reduced by NADH or NADPH (Km = 6.4 and 4.4 microM, respectively). In the presence of free FMN as the electron-accepting substrate, the latter reductant showed a ping-pong Bi-Bi reaction mechanism, whereas utilization of NADH is competitively inhibited by this substrate. This finding suggests that NADPH is the physiological reductant of the enzyme. We also show that YcnD reduces nitro-organic compounds, chromate, and a series of azo dyes. The reduction of azo dyes appears to be mediated by free reduced FMN because the reaction is considerably slower in its absence. Structure determination by X-ray crystallography revealed that YcnD folds into a three layer alpha-beta-alpha sandwich strongly resembling the topology of the NADH oxidase superfamily. Similar to homologous bacterial oxidoreductase, YcnD forms homodimers with an extended dimer interface. The biochemical data and the structure are discussed in light of the putative physiological function of YcnD as an oxidoreductase delivering reduced FMN to enzymes that require the reduced cofactor for activity.
authors.affiliation: Institut für Biochemie, Technische Universität Graz, A-8010 Graz, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Morokutti
authors.forename: Andrzej
authors.initials: A
authors.lastname: Lyskowski
authors.forename: Sonja
authors.initials: S
authors.lastname: Sollner
authors.forename: Eva
authors.initials: E
authors.lastname: Pointner
authors.forename: Teresa B
authors.initials: TB
authors.lastname: Fitzpatrick
authors.forename: Christoph
authors.initials: C
authors.lastname: Kratky
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D005486:Flavin Mononucleotide; D010088:Oxidoreductases