恶臭假单胞菌SJTE-1中转化17β-雌二醇的3-酰基-ACP还原酶的鉴定与功能研究

A new 3-oxoacyl-acyl-carrier-protein reductase identified in Pseudomonas putida SJTE-1 can catalyze the transformation of 17β-estraiol

摘要:

[目的]假单胞菌SJTE-1可高效转化17β-雌二醇，但是催化该转化的酶尚不清楚。本文鉴定了该菌株的一个新的3-酮酰基-ACP还原酶（ANI01589.1），并对其进行了功能研究。[方法]首先，我们克隆了该3-酰基-ACP还原酶的编码基因，在大肠杆菌BL21（DE3）菌株中进行了异源表达；利用金属离子亲和层析法，纯化获得了重组蛋白。体外检测了重组蛋白的活性与酶学性质，并利用高效液相色谱法（HPLC）测定了该酶的催化产物。[结果]3-酮酰基-ACP还原酶可被17β-雌二醇诱导表达，重组蛋白纯化量可达19.6 mg/L。蛋白序列比对结果表明，该蛋白包含短链脱氢酶/还原酶（SDR）的2个共有区域和多个保守残基。该酶以NAD⁺为辅助因子，将17β-雌二醇转化为雌酮；其K_m值为0.071 mmol/L，k_cat值为2.4±0.06/s^-1，5 min内可转化超过95.8%的雌二醇。该酶的最佳反应温度为42℃，最佳pH为8.0。不同二价离子对该酶的活性影响不同，Mg²⁺和Mn²⁺可增强其酶活性。[结论]这一假单胞菌SJTE-1来源的3-酮酰基-ACP还原酶可高效催化17β-雌二醇的转化，该酶可能在该菌株的雌激素代谢过程中起到重要作用。

Abstract:

[Objective] Pseudomonas putida SJTE-1 can degrade 17β-estradiol (E2) efficiently, but the enzymes for the tranformation of E2 in this strain is still unclear. In this work, we identified and characterized a new 3-oxoacyl-acyl-carrier-protein reductase (3-oxoacyl-ACP reductase, ANI01589.1) involved in the E2 degradation. [Methods] We cloned the encoding gene of this 3-oxoacyl-ACP reductase and overexpressed it in Escherichia coli BL21(DE3) strain. We purified the recombinant protein by metal-ion affinity chromatography and characterized its enzymatic activity in vitro. Then we detected the product of this enzymatic reaction with High Performance Liquid Chromatography (HPLC). [Results] The transcription of the 3-oxoacyl-ACP reductase was induced by 17β-estradiol. Protein sequence alignment showed that it contained two consensus regions and the conserved residues of the short-chain dehydrogenase/reductase (SDR), and its structure was similar to that of the 3-oxoacyl-ACP reductase (4fw8.1.A). The recombinant protein was purified with the yield of 19.6 mg per liter culture. This 3-oxoacyl-ACP reductase could convert 17β-estradiol into estrone using NAD⁺ as the cofactor. Its K_m value was 0.071 mmol/L and its k_cat value was 2.4±0.06/s^-1; and the transformation efficiency of this enzyme to 17β-estradiol was over 95.8% in 5 min. Its optimal reaction temperature was 42℃ and the optimal pH was 8.0. Divalent ions had different effect on the enzymatic activity; Mg²⁺ and Mn²⁺ could enhance the enzymatic activity. [Conclusion] The 3-oxoacyl-ACP reductase (ANI01589.1) could catalyze the transformation of 17β-estraiol efficiently and was important for the estrogen metabolism of P. putida SJTE-1.