Mechanistic Characterisation of a Diterpene Synthase for Chryseojoostenes A-E from Chryseobacterium Joostei

A diterpene synthase from Chryseobacterium joostei was characterised and produces the five unique compounds chryseojoostenes A-E. Chryseojoostenes D and E were produced in too low amounts for isolation from the wildtype enzyme, but extensive site-directed mutagenesis resulted in an enzyme variant in which the production of these compounds was enhanced. The biosynthesis of the enzyme products was investigated in detail through a combined experimental and computational approach, indicating a complex hydrogen scrambling during terpene cyclisation and a long-range proton shift towards chryseojoostene E. Density functional theory (DFT) calculations revealed that a similar long range hydrogen shift is involved in the formation of an even fragment ion (m/z 216), characterising the unique chemistry of the chryseojoostene skeleton. Further insights into the cyclisation mechanism were obtained by enzymatic conversion of two substrate analogs with reduced reactivity.