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-X -Cys-X -Cys-X -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.

Mechanistic Insights into Interactions between Bacterial Class I P450 Enzymes and Redox Partners

“…39 Unlike P450sca2 that mainly produced 19 in all positive reactions, the main products of CYP105AS-1 were 20 and 21 in a majority of positive reactions (Tables S12 and S13). Strikingly, a new product (22) was observed when CYP105AS-1 was in companion with either SelFdx0338/SelFdR0978 or SelFdx1499/SelFdR0978 (Figure 2A, trace (ii) and (iii)) compared to RhFRED as a control, 32 which only led to the production of 20 and 21 (Figure 2A, trace (iv)). The structure was determined as R-195 by high-resolution mass spectrometry (C 18 H 24 NO 5 , [M + H] + : obsd 321.1703, calcd 321.1697) (Figure S2) and 1 H NMR analysis 40 (see the Supporting Information).…”

“…Surprisingly, few studies ,− have been carried out to address the above and the following important questions about the functional pairing between prokaryotic Class I P450s and redox partners, either homologously or heterologously: (1) Are there any P450 preferred types of Fdx and FdR in microorganisms? (2) If yes, what are the recognizing factors of the P450-specific Fdx and FdR?…”

Three pairs of surrogate redox partners comparison for Class I cytochrome P450 enzyme activity reconstitution

“…The electron transfer efficiency of RPs was measured by monitoring the increase of reduced cyt c at 550 nm using extinction coefficient of 21,000 M −1 cm −1 49 in a UV-visible spectrophotometer (Varian, UK). The reaction mixture containing 30 μM cyt c , 1 nM FdR, and varying concentrations of ferredoxin (0–40 μM) in 50 mM potassium phosphate buffer (pH 7.4) 28 .…”

Characterization of enzymatic properties of two novel enzymes, 3,4-dihydroxyphenylacetate dioxygenase and 4-hydroxyphenylacetate 3-hydroxylase, from Sulfobacillus acidophilus TPY

“…Escherichia coli BL21(DE3) cells harboring the plasmids pET-32a(+)-hpaB, pET-32a(+)-hpaC and pET-28a(+)-mhpB2 were cultivated at 37 °C in 100 mL of LB medium supplemented with 100 μg/mL ampicillin or 50 μg/mL kanamycin overnight respectively. Expression and purification of recombinant proteins were done according to our previous work [23]. The protein concentration was determined according to Bradford assay with bovine serum albumin as standard [24].…”