Preliminary Identification Of The Active Site Of Steroid 11α-hydtoxylase Gene CYP68J5

Steroidal drugs have unique cyclopentane polyhydrophenanthrene parent nucleus structure,which has important application value in the pharmaceutical industry,and is widely used in anti-inflammatory,anti-allergy,anti-tumor and birth control.The pharmacological activity of steroid drugs depends on the introduction of functional groups at specific sites of steroid parent nucleus,especially hydroxyl groups at C11.Due to the complex molecular structure of steroid compounds and multiple asymmetric centers on the basic skeleton,traditional chemical synthesis methods for the preparation of steroid drugs have disadvantages such as complicated steps,more byproducts and low benefits.At present,microbial transformation methods are usually used in industry to selectively introduce hydroxyl groups into steroid parent nucleus.Aspergillus ochraceus is an important microorganism in industrial steroidal C11α-hydroxylation reaction,but its potential application value is limited by the narrow substrate spectrum.The hydroxylase of filamentous fungi belongs to P450 enzyme,which is a membrane protein.Due to the difficulty of obtaining crystal structure,rational modification based on structure cannot be applied directly.The key gene CYP68J5 involved in the C11α-hydroxylation reaction of Aspergillus ochraceus was identified,and the byproduct of Aspergillus ochraceus conversion to progesterone was catalyzed by the hydroxylase CYP68J5.To improve the specificity of hydroxylase CYP68J5 for progesterone C11α-hydroxylation and broaden the substrate spectrum,the research contents and results of this paper are as follows: 1)By homologous sequence alignment,site-directed mutagenesis of CYP68J5 gene was performed by PCR site-directed mutation technology to determine the amino acid residues at the active site;2)V64F,N66 T and L129 F were identified as the amino acid residues of the active center through the construction of single mutants and steroid transformation experiments,and the highly specific recombinant strain V64 F was obtained;3)Through saturation mutagenesis,V64 and N66 were identified as amino acid residues affecting the transformation of substrates,and the structure of substrates significantly affected the transformation activity;4)It was confirmed that the C13 ethyl group of diketone was the main reason for the difference of conversion activity between D-ethylgonendione and progesterone by using the derivatives with highly similar structure of steroid substrate as probe,which provided the idea for the conversion of D-ethylgonendione and its derivatives by hydroxylase in the future,and for provide ideas for the construction of highly specific mutants.