RESUMO
Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Citosina/metabolismo , DCMP Desaminase/metabolismo , Desoxirribonucleotídeos/metabolismo , Nucleotídeos de Desoxiuracil/biossíntese , Nucleotídeo Desaminases/metabolismo , Sequência de Aminoácidos , Bacillus/química , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vias Biossintéticas , Cristalografia por Raios X , DCMP Desaminase/química , DCMP Desaminase/genética , Cinética , Dados de Sequência Molecular , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/genética , Especificidade por SubstratoRESUMO
The structure of the all-cysteinyl-coordinated D14C variant of [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus has been determined to 1.7 Å resolution from a crystal belonging to space group C222(1) with two types of molecules, A and B, in the asymmetric unit. A and B molecules have different crystal packing and intramolecular disulfide bond conformation. The crystal packing reveals a ß-sheet interaction between A molecules in adjacent asymmetric units, whereas B molecules are packed as monomers in a less rigid position next to the A-A extended ß-sheet dimers. The A molecules contain an intramolecular disulfide bond in a double conformation with 60% occupancy left-handed and 40% occupancy right-handed spiral conformation, whereas B molecules have an intramolecular disulfide bond in a right-handed spiral conformation. The cluster in D14C [4Fe-4S] P. furiosus ferredoxin was chemically oxidized at pH 5.8 to [3Fe-4S]. For purification at pH 8.0, two forms of the protein are obtained. Mass spectrometric analysis shows that the two forms are the D14C [3Fe-4S] P. furiosus ferredoxin monomer and a disulfide-bonded dimer of D14C [3Fe-4S] P. furiosus ferredoxin. When oxidization and purification are carried out at pH 5.8, only the monomer is obtained. The crystal structure of D14C [3Fe-4S] P. furiosus ferredoxin monomer was determined to 2.8 Å resolution from a crystal belonging to space group P2(1)2(1)2(1) with two molecules in the asymmetric unit. The molecules resemble molecule A of D14C [4Fe-4S] P. furiosus ferredoxin and electron density clearly shows the presence of a [3Fe-4S] cluster.
