title: A novel NADPH-dependent reductase of Sulfobacillus acidophilus TPY phenol hydroxylase: expression, characterization, and functional analysis.
abstract: The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X4-Cys-X2-Cys-X29-35-Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP 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. 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. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.
authors.affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Daxue Road 184, Xiamen, 361005, People's Republic of China.|Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen, 361005, People's Republic of China.
authors.forename: Meng
authors.initials: M
authors.lastname: Li
authors.affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Daxue Road 184, Xiamen, 361005, People's Republic of China.|Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen, 361005, People's Republic of China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Guo
authors.affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Daxue Road 184, Xiamen, 361005, People's Republic of China. chenxinhua@tio.org.cn.|Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen, 361005, People's Republic of China. chenxinhua@tio.org.cn.|Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, People's Republic of China. chenxinhua@tio.org.cn.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Chen
mesh_terms: D020816:Amino Acid Motifs; D000068016:Eubacteriales / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D015870:Gene Expression; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D007506:Iron-Sulfur Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C022336:phenol 2-monooxygenase
keywords: Heterologous expression; Multicomponent phenol hydroxylase; NADPH-dependent reductase; Sulfobacillus acidophilus TPY