Dataset Information

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

ABSTRACT: CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

SUBMITTER: Bracco P

PROVIDER: S-EPMC8048783 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

Bracco Paula P Wijma Hein J HJ Nicolai Bastian B Buitrago Jhon Alexander Rodriguez JAR Klünemann Thomas T Vila Agustina A Schrepfer Patrick P Blankenfeldt Wulf W Janssen Dick B DB Schallmey Anett A

Chembiochem : a European journal of chemical biology 20201130 6

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition ...[more]

PMID: 33145893

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Project description:Purpose: In order to identify genes for 11β-hydroxylase CYP5311B2 and associated redox partners CPR and cytochrome b5 in filamentous fungus Absidia orchidis AS3.65 Methods: Strand-specific RNA-seq libraries were constructed for two samples, including (I) Mycelia of A. orchidis treated with mock N, N-dimethylformamide (DMF); (II) Mycelia of A. orchidis treated with 170 mg/L 11-deoxycortisol 21-acetate (RSA) dissolved in N, N-dimethylformamide. For preparation of RNA samples, germinated young mycelia of A. orchidis after 22-24 pre-culture in liquid potato dextrose broth medium were harvested and transferred into a new 30 ml potassium phosphate buffer supplement with either 170 mg/L RSA dissolved in DMF, or an equal volume of DMF as mock. Additional two-hours cultivation was performed to induction of the 11β-hydroxylase gene expression. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The two 150-nt strand-specific paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). The clean reads were used to create a de novo transcriptome assembly using the Trinity pipeline (v2.4.0). The abundance for each of the resulted transcripts was calculated by RSEM (v1.2.0) using the Fragments Per Kilobase per Million (FPKM) method. Results: A total of 10.4 and 10.5 million 150-bp paired-end clean reads were separately generated from the DMF mock and RSA induced samples,and 57549 unique transcripts were assembled from the pooled reads of the two samples using Trinity. The translated sequences of these transcripts were predicted using TransDecoder, and then used as queries to search against the SWISS-PROT and the Pfam databases for functional annotation.A group of 87 translated sequences were found have a typical CYP domain (PF00067), among which,the expression levels of DN10804 and DN10492 separately increased 306- and 48-fold upon RSA induction and identifed as A. orchidis 11α- and 11β- hydroxylase genes. In addition, DN17017 and DN7580 were identifed as the transcripts for the cytochrome P450 reductase and cytochrome b5,respectively. Conclusions: The 11β-hydroxylase system of A. orchidis was deciphered by transcriptome analysis.

Dataset Information

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

Publications

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

Similar Datasets

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