title: Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.
abstract: The formation of H2 O2 by the FAD-dependent L-α-glycerophosphate oxidase (GlpO) is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. (SspGlpO) is similar to the pneumococcal protein (SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO (MpGlpO), having < 20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO that we designate as type II GlpOs. In the present study, the recombinant His-tagged MpGlpO structure is described at an approximate resolution of 2.5 Å, solved by molecular replacement using, as a search model, the Bordetella pertussis protein 3253 (Bp3253), comprising a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of approximately 580 min(-1), whereas Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for L-α-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. The present study provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases, as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.
  
  
  METHODS
  Structural data have been deposited in the Protein Data Bank under accession numbers 4X9M (oxidized) and 4X9N (reduced).
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.
authors: Callia K
authors: CK
authors: Elkhal
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.
authors: Kelsey M
authors: KM
authors: Kean
authors: Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors: Derek
authors: D
authors: Parsonage
authors: Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand.
authors: Somchart
authors: S
authors: Maenpuen
authors: Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Pimchai
authors: P
authors: Chaiyen
authors: Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors: Al
authors: A
authors: Claiborne
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.
authors: P Andrew
authors: PA
authors: Karplus
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001886:Bordetella pertussis / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009177:Mycoplasma pneumoniae / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005993:Glycerolphosphate Dehydrogenase; C098831:glycerol-3-phosphate oxidase
keywords: GlpA; drug design; flavoenzyme; hydride transfer; protein evolution