title: Solution structure of the lipoyl domain of the chimeric dihydrolipoyl dehydrogenase P64K from Neisseria meningitidis.
abstract: The antigenic P64K protein from the pathogenic bacterium Neisseria meningitidis is found in the outer membrane of the cell, and consists of two parts: an 81-residue N-terminal region and a 482-residue C-terminal region. The amino-acid sequence of the N-terminal region is homologous with the lipoyl domains of the dihydrolipoyl acyltransferase (E2) components, and that of the C-terminal region with the dihydrolipoyl dehydrogenase (E3) components, of 2-oxo acid dehydrogenase multienzyme complexes. The two parts are separated by a long linker region, similar to the linker regions in the E2 chains of 2-oxo acid dehydrogenase complexes, and it is likely this region is conformationally flexible. A subgene encoding the P64K lipoyl domain was created and over-expressed in Escherichia coli. The product was capable of post-translational modification by the lipoate protein ligase but not aberrant modification by the biotin protein ligase of E. coli. The solution structure of the apo-domain was determined by means of heteronuclear NMR spectroscopy and found to be a flattened beta barrel composed of two four-stranded antiparallel beta sheets. The lysine residue that becomes lipoylated is in an exposed beta turn that, from a [1H]-15N heteronuclear Overhauser effect experiment, appears to enjoy substantial local motion. This structure of a lipoyl domain derived from a dihydrolipoyl dehydrogenase resembles that of lipoyl domains normally found as part of the dihydrolipoyl acyltransferase component of 2-oxo acid dehydrogenase complexes and will assist in furthering the understanding of its function in a multienzyme complex and in the membrane-bound P64K protein itself.
authors.affiliation: Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK.
authors.forename: K
authors.initials: K
authors.lastname: Tozawa
authors.forename: R W
authors.initials: RW
authors.lastname: Broadhurst
authors.forename: A R
authors.initials: AR
authors.lastname: Raine
authors.forename: C
authors.initials: C
authors.lastname: Fuller
authors.forename: A
authors.initials: A
authors.lastname: Alvarez
authors.forename: G
authors.initials: G
authors.lastname: Guillen
authors.forename: G
authors.initials: G
authors.lastname: Padron
authors.forename: R N
authors.initials: RN
authors.lastname: Perham
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010453:Peptide Synthases / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D012996:Solutions; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase; D010453:Peptide Synthases; C072322:lipoate-protein ligase