| Bacterial multicomponent monooxygenases(BMMOs) are nonheme, dinuclear iron enzymes capable of using molecular oxygen to hydroxylate a variety of organic substrates, thus contributing to the biodegradation and biocatalysis of organic compounds. BMMs usually contain three common components: dinuclear iron hydroxylase, reductase component, and cofactor- less regulatory protein. Although the carboxylate-bridged dinuclear iron center is in the hydroxylase component, many reports have demonstrated that efficient hydroxylation of substrates by BMMs is not possible without both the reductase and regulatory components. The reductase component is usually an FAD- and [2Fe-2S]-containing enzyme that transfers electrons from NAD(P)H to the di- iron sites of the terminal oxygenase via the [2Fe-2S] cluster and flavin prosthetic group. Therefore, reductase is the key enzyme in the electron transfer of BMMOs. Multicomponent phenol hydroxylase is an important member of the BMMO family, it play a big part in the degradation of phenol which is a hazardous pollutant,because it can convert phenol into less toxic intermediates,Sulfobacillus acidophilus strain TPY is a acidophile and moderate thermophile gram-positive bacterium isolated from a deep-sea hydrothermal vent in the Pacific Ocean, it is an. Sulfobacillus acidophilus TPY can completely degrade 100 mg/L of phenol in 40 h at 45°C and p H 1.8. Amino acid sequence homology analyses suggested that five genes(i.e., mhpLMNOP) encode the five components of PH. In order to clarify the function of PH, in the present study, the reductase component(PHR) encoded by the mhpP gene was expressed and purified and characterized in detail.Amino acid sequence alignment analysis revealed that MhpP has an N-terminal ferredoxin- like domain and C-terminal ferredoxin-NADP reductase(FNR)- like domain, as a typical BMMO reductase. Four cysteine residues are conserved in the N-terminus ferredoxin- like domain of MhpP for [2Fe-2S] cluster binding.Motifs for binding the flavin adenine dinucleotide(FAD) and NADPH are conserved in the C-terminal FNR- like domain. MhpP has close relationship with phenol hydroxylase reductases of some phenol-degradation bacterium,but the low sequence identities of MhpP with these enzymes indicated MhpP has novel amino acid sequence.Mhp P was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV/visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. Thin- layer chromatography revealed that Mhp P contains FAD. MhpP can oxidizes NADPH using cytochrome c, potassium ferricyanide, nitro blue tetrazolium as an electron acceptor. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. Four cysteine residues are conserved in the N-terminus ferredoxin- like domain of Mhp P for [2Fe-2S] cluster binding.MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH.MhpP proved itself as a BMMO reductase as an iron-sulfur flavoprotein that utilizes FAD and a [2Fe-2S] cluster as cofactors and oxidize NADPH and transport electrons. This is the first time expression and characterization of a phenol hydroxylase component of Sulfobacillus acidophilus. Investigation about this reductase is helpful for the further study of enzymes in phenol degradation of Sulfobacillus acidophilus... |
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