Enzyme Properties And Preliminary Analysis Of The Active Sites Of 3-ketosteroid-Δ~1-dehydrogenase Homologues Of Arthrobacter Simplex

The dehydrogenation C1,2 of steroids is one of the most valuable reactions in steroid metabolism,which was catalyzed by 3-ketosteroid-Δ1-dehydrogenase(ksdD).Arthrobacter simplex has been widely used in steroid industry because of its good specificity and high reaction efficiency.However,the information of KsdD contained in this strain is not completely clear.In our previous work,five isoenzymes of KsdD(named as KsdD 1,KsdD2,KsdD3,KsdD4 and KsdD5,respectively)were found by sequencing the whole genome of Arthrobacter simplex,and their catalytic activity was confirmed by single gene overexpression and heterologous expression experiments.On this basis,the transcriptional patterns of isozymes,enzymatic properties of important isozymes and key amino acid sites were analyzed.RT-qPCR was used to analyze the transcriptional levels of ksdD in the wild-type strain and kdD overexpression strains.The transcriptional levels of the five genes in the wild-type strains reached the maximum(ksdD4,ksdD3,ksdD5,ksdD1 and ksdD2 in descending order)after AD induction for 12 h,and then the transcription levels of the five ksdD genes decreased with the extension of induction time.The mRNA levels of ksdD in ksdD overexpression strains were significantly higher than that of the wild-type strain.Meanwhile,the mRNA level of other ksdD genes in A.simplex may be affected by the overexpression of single ksdD gene.Five ksdD genes promoters were predicted by bioinformatics method.The recognition sequences of transcription factor kstR that played an important role in regulating steroid metabolism were found in the upstream of open reading frame of ksdD2 and ksdD3 genes.Five ksdD homologues of A.simplex were modeled by using the ksdD1 of Rhodococcus erythropolis SQ1 strain with known structure as the template.Combined with the previous data of KsdD substrate profiles.The Loop structure of protein at the substrate pocket might be an important factor affecting the substrate preference of KsdD.Based on the role of KsdD in vivo,the important KsdD3 and KsdD5 were selected for purification and enzymatic properties study.The optimum temperature and optimum pH of KsdD3 and KsdD5 were 30℃,7.5 and 35℃,7.0,respectively.The two enzymes showed good thermal stability at 20-30 ℃.In addition,The two enzymes showed good pH stability at 7.0-8.5 and 6.0-8.0,separately.Furthermore,they showed strong organic solvent tolerance and good preference for 4-ene-3-oxosteroids.The active site residues of KsdD5 with good application potential were preliminarily analyzed by site-directed mutagenesis and substrate transformation.The results showed that the amino acid residues(Y118,Y355,Y528 and G532)at the substrate binding sites of KsdD5,as well as the amino acid residues at M100 and D117 played a key role.KsdD5 has some unique key amino acid residues,such as A255,R256,G258,V259,L261,F266 and R273.In addition,T312 and T490 play a certain role.The results provide important basic data for understanding the catalytic mechanism of A.simplex KsdD and subsequent molecular modification.