Recently, Angewandte Chemie International Edition (IF:16.6), one of the top journals in the field of chemistry, published online the latest research results of Liao-Bin Dong's group in the School of Traditional Chinese Medicine of the University of China, “Discovery, Structure, and Engineering of a cis-Geranylfarnesyl Diphosphate Synthase”. Dr. Fangru Li (PhD student, class of 2020) and Qingling Wang (MSc student, class of 2021) are the co-first authors of this paper, while Prof. Liao-Bin Dong is the independent corresponding author, and China Pharmaceutical University (CPU) is the only corresponding author.
Isopentenyltransferase catalyzes the sequential condensation reaction between allyl substrates and isopentenyl pyrophosphate to generate chain pyrophosphate precursors, which are subsequently catalyzed by terpene cyclase to form structurally diverse terpene carbocyclic skeletons. Based on differences in the conformation of the double bonds formed by the condensation reaction, isopentenyltransferases are classified into two types: trans and cis. The vast majority of terpene cyclases use trans linear pyrophosphate as a substrate, but some are capable of utilizing cis linear pyrophosphate to produce terpene natural products such as monoterpenes, sesquiterpenes and diterpenes. However, to date, no isopentenyltransferase has been reported to biosynthesize C25 cis-linear pyrophosphate.
Liao-Bin Dong's group discovered and characterized a novel cis-Geranylfarnesyl pyrophosphate synthase, ScGFPPS, from Streptomyces rodenticus, which was able to catalyze the condensation of C5, C10, and C15 pyrophosphate substrates with isoprenyl pyrophosphate to produce three C25 and three C20 cis-pyrophosphate products, respectively (Figure A). The protein crystal structure of ScGFPPS was obtained by X-ray crystallography. Unlike all reported isoprenyltransferases of this family, ScGFPPS showed a completely symmetrical “butterfly” structure (Figure B). Meanwhile, the protein crystal structure of NNPS, an isoprenyltransferase that synthesizes all-cis C20 pyrophosphate products exclusively, was elucidated for the first time (Figure B). Comparison of the protein crystal structures of ScGFPPS and NNPS revealed the key amino acid residue sites affecting the catalytic function. Further, based on the model of two enzyme-substrate complexes, targeted mutagenesis of the differential amino acid residues significantly improved the catalytic activity of ScGFPPS as well as the selectivity to form a single cis-linear pyrophosphate product. This study guided the engineering modification of the enzyme through the comparison of protein crystal structures, facilitated the application of cis-isopentenyltransferases in the biosynthesis of terpene natural products, and promoted the biosynthesis research of complex terpene structures.
The above work was supported by the National Natural Science Foundation of China (82073746), the National Young Talent Program, “Jiangsu Distinguished Professors” and the Program for Dual Top Universities (CPUQNJC22_04).