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Biochim Biophys Acta ; 1844(4): 778-84, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24534646

RESUMO

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation.


Assuntos
Arginina/química , Aspergillus fumigatus/química , Flavinas/química , Proteínas Fúngicas/química , Oxigenases de Função Mista/química , NADP/química , Ornitina/química , Alanina/química , Alanina/metabolismo , Arginina/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Flavinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Hidroxilação , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , NAD/química , NAD/metabolismo , NADP/metabolismo , Ornitina/metabolismo , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Eletricidade Estática , Especificidade por Substrato
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