Journal: Applied and Environmental Microbiology

Article Title: Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium

doi: 10.1128/aem.01753-23

Figure Lengend Snippet: Amino acid sequence alignment of HGD homologs. Sequences of PcHGD1 (protein ID 6344326), PcHGD2 (6344291), and HGD from Trametes versicolor (TV_20432), Gelatoporia subvermispora (GS_117547), Homo sapiens (Q93099), and Pseudomonas putida (Q88E47) are shown. The protein IDs for H. sapiens and P. putida were obtained from the UniProt Knowledgebase (http://beta.uniprot.org), and those for the other species were obtained from the JGI Genome Portal (https://mycocosm.jgi.doe.gov/Phchr4_2/Phchr4_2.home.html). The residues in the red boxes are responsible for active-site non-heme ferric iron coordination and catalytic activity in HGD. The sequences were aligned using ClustalW program (https://www.genome.jp/tools-bin/clustalw). The active site lid is underlined.

Article Snippet: For the experiments, fungal conidia were inoculated in 30 mL of HCLN medium (pH 4.5) in a 300-mL Erlenmeyer flask and incubated at 37°C in stationary culture. . Construction of the gene expression system Full-length P. chrysosporium PcHGD1 and PcHGD2 genes (6344326 and 6344291) with an amino acid sequence identity of 69.7% were PCR amplified using the primer combinations shown in Table S1 and a DNA thermal cycler 2400 (Takara Bio, Otsu, Japan) as follows: initial denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 2 min, annealing at 60°C for 30 s, and extension at 68°C for 30 s. The primer sets were designed using the genomic sequence of P. chrysosporium ( https://mycocosm.jgi.doe.gov/Phchr4_2/Phchr4_2.home.html ).

Techniques: Sequencing, Activity Assay

Journal: Applied and Environmental Microbiology

Article Title: Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium

doi: 10.1128/aem.01753-23

Figure Lengend Snippet: Preparation of recombinant PcHGD1 and PcHGD2 and oxygen consumption during HGD reactions with HGA as a substrate. (A) SDS-PAGE analysis of purified PcHGD1 (lane 1) and PcHGD2 (lane 2). (B) Oxygen consumption during HGD reactions with HGA. Consumption of oxygen was monitored using a Clark O2 electrode. The arrow indicates the addition of a substrate to the reaction mixture.

Article Snippet: For the experiments, fungal conidia were inoculated in 30 mL of HCLN medium (pH 4.5) in a 300-mL Erlenmeyer flask and incubated at 37°C in stationary culture. . Construction of the gene expression system Full-length P. chrysosporium PcHGD1 and PcHGD2 genes (6344326 and 6344291) with an amino acid sequence identity of 69.7% were PCR amplified using the primer combinations shown in Table S1 and a DNA thermal cycler 2400 (Takara Bio, Otsu, Japan) as follows: initial denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 2 min, annealing at 60°C for 30 s, and extension at 68°C for 30 s. The primer sets were designed using the genomic sequence of P. chrysosporium ( https://mycocosm.jgi.doe.gov/Phchr4_2/Phchr4_2.home.html ).

Techniques: Recombinant, SDS Page, Purification

Journal: Applied and Environmental Microbiology

Article Title: Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium

doi: 10.1128/aem.01753-23

Figure Lengend Snippet: Optimal temperature and pH of PcHGD1 and PcHGD2. (A and B) Optimal temperatures for PcHGD1 and PcHGD2 determined using MHQ as the substrate. Enzyme reactions proceeded at temperatures ranging from 20°C to 70°C. (C and D) Optimal pH of PcHGD1 and PcHGD2. Enzyme reactions proceeded over a pH range of 5.5–8.0: in 50 mM MES (pH 5.0–6.5; ●), 50 mM MOPS (pH 6.5–7.0; ■), and 50 mM HEPES (pH 7.0–8.0; ▲). Data are presented as mean ± standard deviation of four independent experiments.

Article Snippet: For the experiments, fungal conidia were inoculated in 30 mL of HCLN medium (pH 4.5) in a 300-mL Erlenmeyer flask and incubated at 37°C in stationary culture. . Construction of the gene expression system Full-length P. chrysosporium PcHGD1 and PcHGD2 genes (6344326 and 6344291) with an amino acid sequence identity of 69.7% were PCR amplified using the primer combinations shown in Table S1 and a DNA thermal cycler 2400 (Takara Bio, Otsu, Japan) as follows: initial denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 2 min, annealing at 60°C for 30 s, and extension at 68°C for 30 s. The primer sets were designed using the genomic sequence of P. chrysosporium ( https://mycocosm.jgi.doe.gov/Phchr4_2/Phchr4_2.home.html ).

Techniques: Standard Deviation

Journal: Applied and Environmental Microbiology

Article Title: Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium

doi: 10.1128/aem.01753-23

Figure Lengend Snippet: Apparent kinetic parameters of PcHGD1 and PcHGD2 for MHQ, DMHQ, and HGA a

Article Snippet: For the experiments, fungal conidia were inoculated in 30 mL of HCLN medium (pH 4.5) in a 300-mL Erlenmeyer flask and incubated at 37°C in stationary culture. . Construction of the gene expression system Full-length P. chrysosporium PcHGD1 and PcHGD2 genes (6344326 and 6344291) with an amino acid sequence identity of 69.7% were PCR amplified using the primer combinations shown in Table S1 and a DNA thermal cycler 2400 (Takara Bio, Otsu, Japan) as follows: initial denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 2 min, annealing at 60°C for 30 s, and extension at 68°C for 30 s. The primer sets were designed using the genomic sequence of P. chrysosporium ( https://mycocosm.jgi.doe.gov/Phchr4_2/Phchr4_2.home.html ).

Techniques: