Structure-activity Relationship Analysis Of Iron-sulfur Clusters And Their Application In Steroid Biotransformation

9α-Hydroxy-4-androstene-3,17-dione(9α-OH-AD)can be used in the synthesis of corticosteroid steroids as an important steroid intermediate.Taking 3-ketosteroid 9α-hydroxylase KSH(composed of oxygenase Ksh A and ferredoxin reductase Ksh B)to catalyze the reaction system of substrate 4-androstene-3,17-dione(4-AD)to produce 9α-OH-AD as the research object,previous studies have shown that Ksh B is a reaction rate-limiting enzyme in the system,so a self-sufficient P450 enzyme-derived reductase PRF was screened.The results showed that PRF had promising NADH reducing activity and 9α-OH-AD conversion rate.The structure-activity relationship of [2Fe-2S] clusters was analyzed and a novel electron transport pathway was proposed.Finally,a high-efficiency catalytic system was introduced into Escherichia coli to construct an engineered strain for steroid biotransformation applications.Ferredoxin(Fdx)is considered a major electron carrier in biological electron transport and acts as an electron donor in the metabolic pathways of many organisms.The integral role of [2Fe-2S] clusters in the electron transfer process was demonstrated by truncation of the Fdx-binding domains in three ferredoxin reductases(Ksh B,TDO,PRF).Then we tried to elucidate the electron transfer efficiencies of the two types of [2Fe-2S] clusters.Based on the experimental data of site-directed mutagenesis of [2Fe-2S] clusters in reductase to realize the mutual conversion of two types of [2Fe-2S] clusters,it is found that plant-type [2Fe-2S]clusters generally have more significant electron transfer efficiency than Rieske type [2Fe-2S] clusters.At the same time,the [2Fe-2S] cluster-binding domains at the C-terminus of Ksh B,TDO,and PRF were further added to the N-terminus,respectively,to construct fusion proteins containing double [2Fe2S] clusters,all of which significantly improved the substrate conversion rate.Comparing the effects of the addition of the two types of [2Fe-2S] clusters,it seemed that the plant-type [2Fe-2S] clusters have a higher assisting role in the electron transport process.After that,we tried to add free Fdxto the multi-enzyme catalytic reaction system in vitro.Three plant-type Fdxs and three Rieske-type Fdxs were screened by sequence alignment and mining.There is a significant improvement of conversion rate compared with that of the catalytic system without free Fdx,and the results also showed that the plant-type Fdx has a higher electron transfer efficiency.The most efficient multi-enzyme catalytic reaction system(PRF+Dm Fdx2+Ksh A)was further selected for protein-protein interaction analysis,and the results showed that free Fdx seemed to create a new electron channel for electrons from reductase to oxygenase.In order to verify the rationality of this novel electron transfer pathway,GST pull-down experiments and ITC assays showed a significant binding force among the three.The results of redox potential measurement showed that ferredoxin Dm Fdx2 has stronger electron transfer ability,and based on the electron bifurcation theory,electrons are more inclined to choose a new electron transfer pathway to complete the electron transfer.Finally,a high-efficiency catalytic system was introduced into Escherichia coli to construct an engineered strain BLA-PD(Ksh A+PRF+Dm Fdx2-BL21).Whole-cell catalytic data showed that the 9α-OH-AD yield was 95.4%,indicating a highly active reductase and efficient electron transfer.The importance of the carrier for steroid transformation and the potential of BLA-PD for the efficient production of 9α-OH-AD in industry.