The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase
The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase
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DOI：
10.1194/jlr.m083949
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发表时间：
2018-06-01
影响因子：
6.5
通讯作者：
Ridlon, Jason M.
中科院分区：
文献类型：
--
作者：
Devendran, Saravanan;Mythen, Sean M.;Ridlon, Jason M.
梭状芽胞杆菌是一种肠道微生物，能够去除皮质醇的侧链，形成11 -羟基雄烯二酮。先前通过RNA-Seq在C. scindens ATCC 35704中发现了一个皮质醇诱导的操纵子（desABCD）。desC基因编码皮质醇20-羟基类固醇脱氢酶（20 α - hsdh）。desD编码一种蛋白质，作为主要促进因子家族的成员，预计作为皮质醇转运蛋白发挥作用。desA和desB基因分别被标注为n端转酮酶和c端转酮酶。我们假设DesAB形成复合物并具有类固醇-17,20-去糖酶活性。我们在pETDuet中克隆了C. scindens ATCC 35704的desA和desB基因，并在大肠杆菌中过表达。经鉴定，纯化的重组DesAB为142 +/- 5.4 kDa的异四聚体。我们开发了一种酶联连续分光光度法来定量类固醇-17,20去糖化酶。这是通过将来自丝状真菌Cochliobolus lunatus的重组17 β - hsdh结合desab依赖的11 β -羟基雄烯二酮的形成与nadph依赖的类固醇17-酮基团的还原来实现的。偶联试验的最佳pH为7.0，以皮质醇为底物的动力学常数k - M为4.96 +/- 0.57 μ M, k（cat）为0.87 +/- 0.076 min（-1）。底物特异性研究表明，rDesAB识别底物与11 β -羟基化无关，但绝对需要17,21-二羟基20-酮类固醇。
Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11 beta-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20-hydroxysteroid dehydrogenase (20 alpha-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 +/- 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11 beta-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17 beta-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were K-m of 4.96 +/- 0.57 mu M and k(cat) of 0.87 +/- 0.076 min(-1). Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11 beta-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.