Customized exogenous ferredoxin functions as an efficient electron carrier

Abstract

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising production yield of 9OHAD (9α-hydroxy4-androstene-3,17-dione) from AD, and further proved the importance of [2Fe–2S] clusters of ferredoxin-oxidoreductase in transferring electrons in steroidal conversion. The results of truncated Fdx domain in all oxidoreductases and mutagenesis data elucidated the indispensable role of [2Fe–2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD (4-androstene-3,17-dione) conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF + Fdx + KshA (KshA, Rieske-type oxygenase of 3-ketosteroid-9-hydroxylase) in the reaction system rather than KshAB complex system was proposed based on analysis of protein–protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier. Graphical Abstract

Directed self-assembly strategy of a DET complex based on the application of artificial electron channeling

“…30 In our previous study, the direct electron transfer (DET) multienzyme complex, involving reductase PRF from Tepidiphilus thermophilus self-sufficient P450 CYP116B46, ferredoxin DmFdx2 from Drosophila melanogaster and oxygenase KshA from Rhodococcus erythropolis, was successfully constructed and exhibited a promising product yield of 9OHAD. 7 The DET is a novel electron transfer pathway based on customized exogenous ferredoxin (Fdx) as an efficient electron carrier (Fig. S1 †).…”

“…† The construction of plasmids, expression and purification of proteins were the same as in our previous study. 7 Analysis of interfacial interactions in the multienzyme complex A three-dimensional (3D) homology model was constructed using the automodel command in Modeller 9.9.2, with the crystal structure of the Rhodococcus rhodochrous 3-ketosteroid-9alpha-hydroxylase 5 monomer (PDB ID: 4QDC, 75.38% sequence identity with KshA) and urate oxidase from Aspergillus flavus (PDB ID: 3PLM, 61.74% sequence identity with DmFdx2) as the templates. The DOPE assessment scores were used to choose the best model.…”

“…In our previous study, we found that the PRF reductase from Tepidiphilus thermophilus self-sufficient P450 CYP116B46 exhibited promising NADPH reduction activity and a higher 9-OHAD yield than KshB. 7 Structural studies have revealed that the oxygenase component KshA is trimeric and the subunit comprises an N-terminal domain harboring the Rieske [2Fe-2S] cluster and a larger catalytic domain harboring the mononuclear Fe. The subunits are arranged head-to-tail within the trimer such that the [2Fe-2S] cluster and the mononuclear iron of adjacent subunits are within 12 Å of each other and comprise the functional unit of the enzyme.…”

“…The 9OHAD yield reached 95.4%, indicating that the new electron transfer pathway was rational and efficient. 7 In general, free enzymes are sensitive and unstable, as well as difficult to recycle and reuse efficiently. [16][17][18] Due to the great demand for more robust catalysts from industry, tremendous efforts have been made to improve the stability of enzymes.…”

Driving the conversion of phytosterol to steroid synthons in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide

“…The catabolism of sterols is a process of oxidative degradation with generation of multiple reducing equivalent. For instance, approximately 17 equivalents nicotinamide adenine dinucleotide (NADH) and 9 equivalents avin adenine dinucleotide (FADH 2 ) can be generated, in theory, during the conversion process from sitosterol to 9-OHAD [9,12,16], if the formed metabolites acetyl-coenzyme A (CoA) and propionyl-CoA can be completely degraded to CO 2 and H 2 O [13,17] as well as the key 9α-hydroxylase is functionalized by FAD transporting electrons from ferredoxin reductase KshB to terminal oxygenase KshA in the 9α-hydroxylation of steroids [18,19] (Fig. 1).…”