Geranylgeranyl diphosphate phosphatase enzyme and gene of plaunotol biosynthetic pathway in croton stellatopilosus ohba

Abstract

The whole leaves and callus cultures of Croton stellatopilosus Ohba could incorporate [l-³H]geranylgeraniol into plaunotol. Enzymological studies showed that geranylgeranyl diphosphate phosphatase (GGDP phosphatase) activity was present in cell- free extracts of the leaves and of chloroplasts. The crude enzyme extract could be separated into two peaks of GGDP phosphatase acitivities (PI and PII) by gel filtration. Purification and characterization of both enzyme peaks revealed that PI was a tetrameric enzyme of 232 kD with its subunit size of 58 kD. It showed optimum pH of 6.0-6.5, an apparent ATm of 0.2 mM, and a Fmax of 277.8 pkat/mg. PII was a monomeric enzyme of 30-34 kD with an apparent ^m value of 0.1 mM, Fmax of 7.5 nkat/mg and exhibited substrate inhibition. Both PI and PII appeared to be membrane-bound enzymes and their activities could be inhibited by Mo2+. Both preferred GGDP as their substrate rather than geranyl diphosphate or farnesyl diphosphate. Study on the catalysis of the reaction revealed that GGOH was formed from GGDP by two successive monodephosphorylations, rather than a one-step diphosphate dephosphorylation. Cloning of the phosphatase genes from C. stellatopilosus leaves, based on the available information of prenyl diphosphate phosphatase in Swiss-Prot database, was also performed using E. coll as expression system. The results showed that the encoded protein had its molecular weight of 33.6 kD and could exhibit GGDP phosphatase activity. The expressed phosphatase also showed its process of catalytic dephosphorylation by using two sequential steps of monophosphate dephosphorylation from GGDP to GGMP and to GGOH.