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( Pradeep Subedi ),( Ki-hwa Kim ),( Young-soo Hong ),( Joo-ho Lee ),( Tae-jin Oh ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.3
Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners’ putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.
Pradeep Adhikari,Sthir Babu Subedi,Suresh Rai 제주대학교 교육과학연구소 2017 교육과학연구 Vol.19 No.1
In this global era, developing and developed countries in the world are facing numerous challenges of environmental protection, resource conservation, sustainable development, climate change adaptation, and global warming mitigation. As such, environmental education (EE) has been necessary in school education for the production of conscious, knowledgeable, and skilled young generations to resolve environmental problems. This study is an attempt to appraise the status of EE in school education of Nepal. We collected the earlier and current curriculums and textbooks of the Basic Education (Grade 1-8) and Secondary Education (Grade 9-10) and determined the subject matters of EE providing in school. We found the curriculum and textbooks deal present environmental issues such as pollution control, prevention from natural disasters, conservation of the ecosystem, natural resources, and wildlife, in both earlier and current curriculum. In addition, the current curriculum included global Environmental issues such as climate change, global warming, and sustainable development. The secondary information was collected to check the learning achievement of EE, which revealed students are knowledgeable, aware, and participates in environment protection activities. Proper implementation of designed curriculum and involvement of student’s in environment protection activities are required to accomplish the desired goal of EE.
Hemraj Rimal,Pradeep Subedi,김기화,박현,이준혁,오태진 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.11
The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated KD value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min−1 nmol−1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.
( Ki-hwa Kim ),( Hackwon Do ),( Chang Woo Lee ),( Pradeep Subedi ),( Mieyoung Choi ),( Yewon Nam ),( Jun Hyuck Lee ),( Tae-jin Oh ) 한국미생물생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.3
Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 A to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.