Exploring the catalytic cascade of cembranoid biosynthesis by combination of genetic engineering and molecular simulations


Schrepfer P., Ugur I., Klumpe S., Loll B., Kaila V. R. , Bruck T.

COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, vol.18, pp.1819-1829, 2020 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 18
  • Publication Date: 2020
  • Doi Number: 10.1016/j.csbj.2020.06.030
  • Title of Journal : COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL
  • Page Numbers: pp.1819-1829
  • Keywords: Terpene cyclization, QM/MM, Oxy-functionalization, Sustainable biocatalysis, Molecular dynamics, BORNYL DIPHOSPHATE SYNTHASE, TAXADIENE SYNTHASE, STRUCTURAL BASIS, BASIS-SETS, CYCLIZATION, DYNAMICS, OPTIMIZATION, PRECURSOR, EFFICIENT

Abstract

While chemical steps involved in bioactive cembranoid biosynthesis have been examined, the corresponding enzymatic mechanisms leading to their formation remain elusive. In the tobacco plant, Nicotiana tabacum, a putative cembratriene-ol synthase (CBTS) initiates the catalytic cascade that lead to the biosynthesis of cembratriene-4,6-diols, which displays antibacterial- and anti-proliferative activities. We report here on structural homology models, functional studies, and mechanistic explorations of this enzyme using a combination of biosynthetic and computational methods. This approach guided us to develop an efficient de novo production of five bioactive non- and monohydroxylated cembranoids. Our homology models in combination with quantum and classical simulations suggested putative principles of the CBTS catalytic cycle, and provided a possible rationale for the formation of premature olefinic side products. Moreover, the functional reconstruction of a N. tabacum-derived class II P450 with a cognate CPR, obtained by transcriptome mining provided for production of bioactive cembratriene-4,6-diols. Our combined findings provide mechanistic insights into cembranoid biosynthesis, and a basis for the sustainable industrial production of highly valuable bioactive cembranoids. (C) 2020 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.