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

Computational and Structural Biotechnology Journal
Patrick SchrepferThomas Brück

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 produ...Continue Reading

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Citations

Feb 4, 2021·Microbial Cell Factories·Yu ZhangHongbo Zhang

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Datasets Mentioned

BETA
AF166332.1
AAS46038.1
AAD47832.1

Methods Mentioned

BETA
size exclusion chromatography
column chromatography
nuclear magnetic resonance
NMR
X-ray

Software Mentioned

def2
TBLASTN
LigPlot
AutoSmiles
NAMD2
CHARMM27
YASARA Structure
TURBOMOLE
YASARA
HHpred

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