Ancestral diterpene cyclases show increased thermostability and substrate acceptance. (16th November 2018)

• Record Type: Journal Article
• Authors: Hendrikse, Natalie M.; Charpentier, Gwenaëlle; Nordling, Erik; Syrén, Per‐Olof
• Is Part Of: FEBS journal. Volume 285:Number 24(2018)
• Page Start: 4660
• Page End: 4673
• Publication Date: 2018-11-16
• Subjects: ancestral sequence reconstruction -- diterpene cyclase -- spiroviolene synthase -- protein stability -- promiscuity; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.14686
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli . Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7–13 °C. Ancestral spiroviolene synthases catalyze cyclization of the natural C20 ‐substrate geranylgeranyl diphosphate (GGPP) and also accept C15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate‐rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., k cat / K M for GGPP over k cat / K M for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism. Abstract : Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display enhanced stability, activity, and promiscuity. We observed a shift in temperature optimum of catalysis compared to the modern enzyme as well as increased yield of soluble protein upon expression in Escherichia coli . Our findings highlight the high potential of applying ancestral sequence reconstruction on biocatalysts within secondary metabolism.