title: GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans.
abstract: Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA (cellulase synthase-like protein). In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective, the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.
authors.affiliation: School of Biological Science, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, U.K.
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Chaplin
authors.affiliation: Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.
authors.forename: Marloes L C
authors.initials: ML
authors.lastname: Petrus
authors.affiliation: Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.
authors.forename: Giulia
authors.initials: G
authors.lastname: Mangiameli
authors.affiliation: School of Biological Science, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, U.K.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hough
authors.affiliation: School of Biological Science, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, U.K.
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.affiliation: School of Biological Science, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, U.K.
authors.forename: Peter
authors.initials: P
authors.lastname: Nicholls
authors.affiliation: Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.
authors.forename: Dennis
authors.initials: D
authors.lastname: Claessen
authors.affiliation: Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands jworrall@essex.ac.uk vijgenbo@biology.leidenuniv.nl.
authors.forename: Erik
authors.initials: E
authors.lastname: Vijgenboom
authors.affiliation: School of Biological Science, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, U.K. jworrall@essex.ac.uk vijgenbo@biology.leidenuniv.nl.
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Worrall
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D025301:Hyphae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D008958:Models, Molecular; D009024:Morphogenesis; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D048170:Streptomyces lividans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D003300:Copper; D010088:Oxidoreductases; C024748:copper oxidase
keywords: X-ray structure; cuproenzyme; development; glycan biotechnology; protein radical; streptomycetes