Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium

Abstract

The activity of a self-sufficient cytochrome P450 enzyme CYP505D6 from the lignin-degrading basidiomycete was characterized. Recombinant CYP505D6 was produced in and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as substrates. Hydroxylation occurred at ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant, and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid analyzed. Compared with the wild-type CYP505D6, its V51Y variant generated fewer products hydroxylated at ω-4 to ω-6 positions. The products generated by the wild-type CYP102A1 were hydroxylated at ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regio-specificity of fatty acid hydroxylation, at least that at ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry. is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in the current study, CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at ω-1 to ω-7 or ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than that of the known fused proteins CYP102A1 and CYP505A1. Substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

CYP505E3: A Novel Self‐Sufficient ω‐7 In‐Chain Hydroxylase

“…Recently, Sakai et al. reported that CYP505D6, a self‐sufficient P450 from the white‐rot fungus Phanerochaete chrysosporium , preferably hydroxylates 1‐dodecanol and dodecanoic acid at the sub‐terminal positions (ω‐1 to ω‐3), but also yielded the in‐chain hydroxylated products (ω‐4 to ω‐7) as minor products . However, to date, no catalysts have been described for the regioselective in‐chain oxyfunctionalization of n ‐alkanes, where no directing functional group is available.…”

Oxygen radical based on non-thermal atmospheric pressure plasma alleviates lignin-derived phenolic toxicity in yeast

“…Vanillin solutions and the hydrolysates (500 μL) were lyophilized, trimethylsilylated using 50 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (Wako Pure Chemical Industries), and analysed using gas chromatography-mass spectrometry (GC-MS; GCMS-QP2010; Shimadzu, Kyoto, Japan) on a system equipped with a J&W DB-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm thickness; Agilent Technologies, Santa Clara, CA) [62]. Glycine (at a final concentration of 50 μM) was used an internal standard for quantitative analysis using GC-MS. We determined the reducing sugar content in the hydrolysates obtained from alkaline-pretreated rice straw with or without oxygenradical and cellulase treatments following filter sterilization.…”

Isolation and Characterization of Dominant Fungi from Rhizospheric Soil of Saussurea obvallata (DC.) Edgew. (Brahma Kamal) of the Indian Himalayan Region

“…The Phanerochaete chrysosporium commonly known as white rot fungi 16 is well known for digestion of fragrant poisons, lignin and lipids and remediation of various pollutants such as PAH, heavy metals and dyes 17 . Phanerochaete chrysosporium is considered an uncommon organism for its capacity to depolymerize and change lignin, cellulose and hemicellulose, in comparison with different basidiomycetes 18 .…”