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Ancestral diterpene cyclases show increased thermostability and substrate acceptance
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi, Ytbehandlingsteknik. KTH, Centra, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.ORCID-id: 0000-0001-8644-3408
KTH, Centra, Science for Life Laboratory, SciLifeLab. ESCOM, 1 Allee Reseau Jean Marie Buckmaster, F-60200 Compiegne, France..
Swedish Orphan Biovitrum AB, Stockholm, Sweden..
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi, Ytbehandlingsteknik. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.ORCID-id: 0000-0002-4066-2776
2018 (Engelska)Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, nr 24, s. 4660-4673Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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 degrees C. Ancestral spiroviolene synthases catalyze cyclization of the natural C-20-substrate geranylgeranyl diphosphate (GGPP) and also accept C-15 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.

Ort, förlag, år, upplaga, sidor
Wiley-VCH Verlagsgesellschaft, 2018. Vol. 285, nr 24, s. 4660-4673
Nyckelord [en]
ancestral sequence reconstruction, diterpene cyclase, spiroviolene synthase, protein stability, promiscuity
Nationell ämneskategori
Kemi
Identifikatorer
URN: urn:nbn:se:kth:diva-240741DOI: 10.1111/febs.14686ISI: 000453570200010PubMedID: 30369053Scopus ID: 2-s2.0-85056620593OAI: oai:DiVA.org:kth-240741DiVA, id: diva2:1274841
Forskningsfinansiär
VINNOVA, 2016-03344Stiftelsen för strategisk forskning (SSF), ID16-0036Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Anmärkning

QC 20190103

Tillgänglig från: 2019-01-03 Skapad: 2019-01-03 Senast uppdaterad: 2019-01-07Bibliografiskt granskad

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Hendrikse, NatalieSyrén, Per-Olof

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Hendrikse, NatalieCharpentier, GwenaelleSyrén, Per-Olof
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