title: Factors involved in the assembly of a functional molybdopyranopterin center in recombinant Comamonas acidovorans xanthine dehydrogenase.
abstract: Previous work from this laboratory has shown that the spectral and functional properties of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans show some similarities to those of the well-characterized eukaryotic enzymes isolated from bovine milk and from chicken liver [Xiang, Q. & Edmondson, D.E. (1996) Biochemistry35, 5441-5450]. Therefore, this system was chosen to study the factors involved in the expression of functional recombinant enzyme in Escherichia coli to provide insights into the assembly of the functional Mo-pyranopterin center. Genes xdhA and xdhB (encoding the two known subunits of the native enzyme) and putative genes xprA and ssuABC were sequenced. Heterologous expression of the xdhAB genes in E. coli JM109(DE3) produced active enzyme. The Mo content was 0.11-0.16 mol per alphabeta protomer, while the Fe and FAD levels were at stoichiometries similar to that of the native enzyme. The XDH activity increased sixfold when the culture was grown under conditions of low aeration (6 L.min-1) as compared with high aeration (12 L.min-1). Co-expression of the xdhAB genes with the Pseudomonas aeruginosa PA1522 (xdhC) gene increased the level of Mo incorporated into the expressed enzyme to a 1 : 1 stoichiometry. Under these conditions, high levels of functional protein (2.284 U.mg-1 and 8.039 mg.L-1 of culture) were obtained independently of the level of culture aeration. Therefore, the assembly of a functional Mo-pyranopterin center in XDH requires the presence of a functional xdhC gene product. The purified, recombinant XDH shows spectral and kinetic properties identical to those of the native enzyme.
authors: Department of Chemistry, Emory University, Atlanta, GA 30322, USA.
authors: Nikolai V
authors: NV
authors: Ivanov
authors: Frantisek
authors: F
authors: Hubálek
authors: Manuela
authors: M
authors: Trani
authors: Dale E
authors: DE
authors: Edmondson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D020585:Comamonas / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008982:Molybdenum / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011621:Pteridines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014968:Xanthine Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D011621:Pteridines; D011994:Recombinant Proteins; D008982:Molybdenum; D014968:Xanthine Dehydrogenase