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.
Highlights
Cytochromes P450 (CYPs) belong to one of the largest enzyme superfamilies, which is widely distributed in all living organisms like bacteria, fungi, plants and animals [1,2]
The phylogenetic tree obtained with MEGA X revealed that P450-T3 formed a cluster with other members of the CYP109C subfamily, which were identified in S. cellulosum (Supplementary Figure S1), suggesting that P450-T3 belongs to the CYP109C subfamily
Multiple amino acid sequence alignments of P450-T3 with its closest homologs reveal the presence of the three conserved domains of cytochrome P450 (Figure 1)
Summary
Cytochromes P450 (CYPs) belong to one of the largest enzyme superfamilies, which is widely distributed in all living organisms like bacteria, fungi, plants and animals [1,2]. They play a crucial role in life by catalyzing more than 20 types of reactions in regio- and stereoselective manners, such as hydroxylation, dealkylation, epoxidation, oxidation, dehalogenation, dehydrogenation and Catalysts 2020, 10, 1083; doi:10.3390/catal10091083 www.mdpi.com/journal/catalysts. Substrates of heat resistant P450 (usually organic compounds in nature) can interact with enzymes in the solvent phase to be converted into diffusion products in the water phase. The first known thermostable P450 (CYP119) was found in the archaea Sulfolobus solfataricus [9]
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