RESUMO
Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.
Assuntos
Oxirredutases/química , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Estabilidade Enzimática , Dados de Sequência Molecular , Molibdênio , Oxirredutases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/genética , Relação Estrutura-AtividadeRESUMO
The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.
Assuntos
Adenosina/análogos & derivados , Coenzimas , Óxidos N-Cíclicos/metabolismo , Proteínas Ferro-Enxofre , Oxirredutases/genética , Rhodobacter sphaeroides/enzimologia , Adenosina/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Guanina/química , Metaloproteínas/química , Molibdênio/química , Cofatores de Molibdênio , Mutagênese Sítio-Dirigida/genética , Oxirredutases/química , Engenharia de Proteínas , Pteridinas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The dsrA gene encoding the molybdoenzyme dimethyl sulfoxide reductase was isolated by screening phagemid libraries containing restriction fragments of Rhodobacter sphaeroides f. sp. denitrificans genomic DNA with a pool of degenerate oligonucleotides. The encoded 822 amino-acid protein includes a 42 amino-acid periplasmic signal sequence that is cleaved during activation of the enzyme. Both forms of the protein were heterologously expressed in inactive states in E. coli and identified by Western blot analysis.
Assuntos
Proteínas Ferro-Enxofre , Oxirredutases/biossíntese , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Genes Bacterianos , Vetores Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredutases/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Rhodobacter sphaeroides/genéticaRESUMO
Chemical analysis of dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans has shown that its molybdenum center contains two molybdopterin guanine dinucleotide molecules and a single atom of molybdenum. The enzyme, which exists as a monomer of 86 kDa, was shown to contain 1 mol of molybdenum, 4 mol of organic phosphate, and 2 mol of guanine per mole of protein. In addition, the relative yield of Form A, a fluorescent derivative of molybdopterin, was twice that obtained from sulfite oxidase, a protein which contains a single molybdopterin per molybdenum. These findings correlate with the recent report of the presence of two molybdopterin ligands in the tungsten cofactor of aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus, providing the first example of a bis(pterin)molybdenum cofactor and extending this structural motif to the molybdopterin dinucleotide enzymes.
