Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization

Thumbnail Image
Date
2014-07-01
Authors
Jackson, Alana
Hershey, David
Chesnut, Taylor
Xu, Meimei
Major Professor
Advisor
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract

It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral entkaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, we applied a synthetic biology approach to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a “splicing” error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, we can assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally novel enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway.

Series Number
Journal Issue
Is Version Of
Versions
Series
Academic or Administrative Unit
Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of
Type
article
Comments

This is the author’s version of a work that was accepted for publication in Photochemistry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Phytochemistry, VOL 103, 2014, doi: 10.1016/j.phytochem.2014.04.005.

Rights Statement
Copyright
Wed Jan 01 00:00:00 UTC 2014
Funding
DOI
Supplemental Resources
Collections