A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Abstract

Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.

Human Cytochrome P450 1, 2, 3 Families as Pharmacogenes with Emphases on Their Antimalarial and Antituberculosis Drugs and Prevalent African Alleles

“…Enzyme sequences within these families contain about 400–500 amino acids [ 28 , 37 ], and those that share ~40% sequence identity are grouped in the same family, while those sharing over 55% sequence homology are subgrouped together into the same sub-family [ 28 , 38 ]. According to [ 39 ] there are three conserved short regions/sequence motifs throughout the CYP superfamily. The first conserved region is the (A/G)XXXT which is located in helix I [ 37 , 39 ].…”

“…According to [ 39 ] there are three conserved short regions/sequence motifs throughout the CYP superfamily. The first conserved region is the (A/G)XXXT which is located in helix I [ 37 , 39 ]. This is the oxygen binding and activation motif that contains the highly conserved threonine which plays an important role in the third step of the catalytic cycle of CYP enzymes [ 39 ].…”

Effect of Temperature on Coenzymes Associated with Cytochrome P450

“…This has given it a wide range of ability of catalysis that enabled it to alter and manipulate the region and stereo structure of both natural products and xenobiotics substrate. Some of these processes range from hydroxylation, dealkylation, epoxidation, oxidation, dehalogenation, dehydrogenation and reduction (Nguyen et al, 2020). Hence cytochrome P450 monooxygenases have shown promiscuity for a wide range of substrates, thanks to the auxiliary redox partners such as flavoenzymes or iron-sulfur proteins that are often co-expressed to obtain electrons from N.A.D.…”

Analysis of microbial communities in Binh Chau hot spring through metagenomic DNA sequencing

“…Binh Chau hot spring locates in Bung Rieng commune, Xuyen Moc district, Ba Ria -Vung Tau province, which is known as the second hottest hot spring in Vietnam with the temperature of hot spot is around 82°C. This is potential sources for exploiting of thermophiles producing novel thermostable enzymes as well as biocatalytic compounds Nguyen et al, 2020). Similar to other hot springs, challenges in implementing microbial consortia in Binh Chau hot spring have been found due to the naturally low-cell density, and/ or very difficulty in sampling.…”