Study on the catalytic mechanism of triterpene C-29 carboxylases from Tripterygium wilfordii based on directed evolution
Celastrol and wilforlide A are the main active triterpenoids of the traditional Chinese medicine Lei Gong Teng,which have anti-tumour,anti-inflammatory and immunosuppressive activities,and are the material basis for the clinical efficacy of Lei Gong Teng-related Chinese medicinal preparations.By analysing the biosynthetic pathway of active ingredients,optimizing genetic elements and utilizing"cell factory"to produce triterpenoids heterologously will be an effective way to obtain from Tripterygium wilfordii in the future in a"low-cost and high-efficient"manner.CYP712Ks are the first cytochrome P450s involved in the skeleton modification of friedelane-type and oleanane-type triterpenoids in T.wilfordii,and they can catalyse the generation of polpunonic acid from friedelin and 3-epi-katonic acid from β-amyrin by carboxylation at C-29 position.In this study,four multifunctional TwCYP712Kl/2/3/5 were used to clarify the catalytic function and substrate selection preference using in vivo functional characterization in Saccharomyces cerevisiae.The spatial structure of the protein-substrate binding was clarified through homology modeling and molecular docking,and then the differential amino acids in the active pocket of the protein were mutated to clarify the crucial amino acids determining the catalytic function and the selection of substrate structure.A total of 63 mutant elements were constructed,and the amino acid sites affecting the carboxylation function of TwCYP712Ks were analyzed.In particular,the key amino acids affecting the substrate selectivity of TwCYP712K2 towards oleanane-type and friedelane-type triterpenoids were revealed and the TwCYP712K2F127I and TwCYP712K2A227T mutants would result in a reversal of the product ratio.In conclusion,four proteins of TwCYP712Ks were semi-rationally designed by homologous protein alignment and mutual mutation to elucidate multiple amino acid sites determining the catalytic function of the proteins,and a series of activity-enhancing or altering mutants were obtained,which provide abundant catalytic elements for the biosynthesis of active triterpenoids from T.wilfordii,and the mechanism of carboxylation in the C-29 position was initially elucidated.
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