| Hydroxysteroid dehydrogenase(HSDH) plays an important role in the process of bile acid converstion, steroid hormone, cholesterol metabolism, non steroidal aldehyde, ketone and carbonyl reduction and had great application potential in industrial biological catalysis. In this paper, taking Clostridium absonum 7?-HSDH(CA 7?-HSDH) as the research object, the coenzyme binding site of CA 7?-HSDH was modificated by genetic engineering. The interaction between CA 7?-HSDH and cofactor was analysised by molecular dynamics simulation, to clarify the function of key cofactor binding sites in CA 7?-HSDH.The main research content and results are as follows:(1) This paper explored the expression of CA 7?-HSDH about pTWIN1 vector on the base of the GST-fusion heterologous expression. The CA 7?-HSDH /pTWIN1 expression system was successfully constructed and CA 7?-HSDH had a good activity. The method can reduce the cost of CA 7?-HSDH, and provide a certain basis for industrial application.(2) Based on the three-dimensional structure, PCR was applied to modified the cofactor binding sites in CA 7?-HSDH and got T15 A, R16 A, R38 A, R194 A, R16A/R194 A, R38A/R194 A, T15A/R16A/R194 A. The results are as follow:(1)R16A and R194 A showed high activity. Compared with WT, activities were increased by 1.8 and 4.8 times, respectively. Both kcat and Km increased, R16 A kcat/Km decreased by 16.2%, and R194 A kcat/Km increased 3 times.(2)Compared with WT, the activity of T15A?R16A/R194 A and T15A/R16A/R194 A were decreased by 26.8%, 55.2%, 75.9%, respectively.Theirs kcat/Km decreased by 81.8%, 43.4%, 84.6% resulting from reduced kcat and increased Km.(3)The catalytic activity of R38 A and R38A/R194 A were not detected, suggesting the key role of R38.(4)All the active mutants' Km and single point mutants' kcat were increased, suggested that the functions of T15?R16?R194 were in the enzyme and coenzyme affinity and catalytic speed, further, T15 in affinity and R194 in speed had outstanding contributions.(3) Using MD simulation technology, the details between mutants and cofactor were studied.(1)The free binding energies results show that mutants' free binding energies about NADP+ compared with the WT significantly increased. Such results were corresponding to the results of activity assay.(2)Structure analysis of R38 A and R38A/R194 A indicated the interaction pattern changed because of cation-? interaction disappeared, the reason for inactivation of the mutants was the destruction of the combination between cofactor and CA 7?-HSDH. On the contrary, T15 A, R16 A, R194 A, R16A/R194 A, T15A/R16A/R194 A remained biological function because of cation-? interaction existenced. These results indicated that R38 played a important role in the binding process of CA 7?-HSDH and cofactor.(3)The hydrogen bond interaction with phosphate group disappeared because of the mutant of 15 th,16th,194 th residue, arginine to alanine, indicating that hydrogen bond directly affects the affinity between CA 7?-HSDH and cofactor, and affinity with the optimum range. In the range, the appropriate affinity could increase the catalytic activity and catalytic efficiency; In addition, The hydrogen bond interaction with pyrophosphate brige disappeared because of the mutant of 194 th residue, arginine to alanine, which leaded to increase the flexibility of pyrophosphate, accelerate the dissociation of nicotinamide ring, and accelerate the reaction speed.(4)The catalytic efficiency decreased with the decreasing amount of charge in R16A/R194 A and T15A/R16A/R194A; On the other hand, in the two mutants, the existence of the polar interaction between S39 and 2'- phosphate, was likely to offset the decline of affinity. That indicated that sufficient power and the suitable affinity were prerequisites to get good activity and high catalytic efficiency enzymes. |
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