RESUMO
APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.
Assuntos
Proteínas de Bactérias/química , Cisteína/química , Proteínas Ferro-Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cisteína/genética , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Ferro-Enxofre/genética , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.
Assuntos
Quitinases/genética , Quitinases/metabolismo , Magnaporthe , Controle Biológico de Vetores , Poaceae/microbiologia , Stenotrophomonas maltophilia/enzimologia , Sequência de Aminoácidos , Burkholderia cepacia/enzimologia , Burkholderia cepacia/genética , Quitina/metabolismo , Clonagem Molecular , Magnaporthe/crescimento & desenvolvimento , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Stenotrophomonas maltophilia/genéticaRESUMO
Sulfur is essential for life. Its oxidation state is in constant flux as it circulates through the global sulfur cycle. Plants play a key role in the cycle since they are primary producers of organic sulfur compounds. They are able to couple photosynthesis to the reduction of sulfate, assimilation into cysteine, and further metabolism into methionine, glutathione, and many other compounds. The activity of the sulfur assimilation pathway responds dynamically to changes in sulfur supply and to environmental conditions that alter the need for reduced sulfur. Molecular genetic analysis has allowed many of the enzymes and regulatory mechanisms involved in the process to be defined. This review focuses on recent advances in the field of plant sulfur metabolism. It also emphasizes areas about which little is known, including transport and recycling/degradation of sulfur compounds.
