RESUMEN
The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with addition of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation.
Asunto(s)
Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Extractos Celulares , Clostridium/enzimología , Clostridium/genética , Activación Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Hidrógeno/metabolismo , Hidrogenasas/química , Hidrogenasas/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
A gene-shuffling technique was identified, optimized and used to generate diverse libraries of recombinant [FeFe]-hydrogenases. Six native [FeFe]-hydrogenase genes from species of Clostridia were first cloned and separately expressed in Escherichia coli concomitantly with the assembly proteins required for [FeFe]-hydrogenase maturation. All enzymes, with the exception of C. thermocellum HydA, exhibited significant activity when expressed. Single-stranded DNA fragments from genes encoding the two most active [FeFe]-hydrogenases were used to optimize a gene-shuffling protocol and generate recombinant enzyme libraries. Random sampling demonstrates that several shuffled products are active. This represents the first successful application of gene-shuffling using hydrogenases. Moreover, we demonstrate that a single set of [FeFe]-hydrogenase maturation proteins is sufficient for the heterologous assembly of the bioinorganic active site of several native and shuffled [FeFe]-hydrogenases.