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

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.