Page 121 - PC2019 Program & Proceedings

PLANT CANADA 2019

S27. Parallel branch pathways have evolved for assembly of major monoterpenoid indole alkaloids
with opposite optical rotations in Catharanthus roseus
2
Williams, D. ; Y. Qu ; V. De Luca 1
*1
1 Brock University
2 University of New Brunswick

Investigations into the biosynthesis of monoterpenoid indole alkaloids (MIAs) in the medicinal
plant, Catharanthus roseus, have led to discoveries of multiple branch pathways leading to their unique
and diverse chemistries. MIAs have complex and unique ring structures that cannot be reproduced by
chemical synthesis, and many have medicinally relevant biological activities. The aspidosperma MIAs are
the most prevalent root-specific alkaloids in C. roseus, and it was previously thought that these MIAs
were derived through various decorations of the tabersonine backbone. Recently, it was shown that
separate hydrolases in C. roseusare responsible for the production of two aspidosperma-type MIAs with
opposite optical rotations, (-)-tabersonine and (+)-vincadifformine, from a common intermediate (PNAS
2018, 115(12):3180-3185; Plant J 2019, 97(2):257-266). The present study shows that separate
hydrolases, hydroxylases and O-acetytransferases catalyse parallel pathways in the formation of (-)-
tabersonine and (+)-vincadifformine derivatives such as (-)-19-O-acetylhörhammericine and (+)-19-O-
acetylvincadifformine, respectively (Plant J 2019, doi: 10.1111/tpj.14346). The enantiomeric-specificity
of these enzymes for their respective (-)- and (+)-substrates sheds new light on the evolution of
specialized metabolism and on the importance of taking stereochemistry into consideration in the
discovery of new pathways.

Danielle Williams (dw15qi@brocku.ca)

S28. Towards understanding the basis of substrate specificity in a newly characterized class of plant
acyl-ACP thioesterases that produce high-value medium-chain fatty acids
*
Kalinger, R. ; I. Pulsifer; O. Rowland
Carleton University

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids,
contribute to much of the fatty acid diversity in plants. ACYL LIPID THIOESTERASES (ALTs), a novel
class of plastid-localized thioesterases occurring in all plant taxa, generate medium-chain (C6-C14) fatty
and β–keto fatty acids as secondary metabolites. Their volatile products likely serve to defend against
predatory insects and pathogens. Medium-chain fatty acids are also used industrially to manufacture
insecticides, pharmaceuticals, and biofuels. We investigated the catalytic diversity of ALT enzymes by
expressing 15 ALTs from monocots, eudicots, a lycophyte, a green microalga, and Ginkgo biloba in
Escherichia coli. Based on their substrate preferences in terms of chain length and oxidation state, the
chosen ALTs could be classified into four catalytic groups comprising enzymes from diverse species and
taxa. Structure-based phylogenetic analyses using three-dimensional models of ALTs revealed unique
tertiary structural features of ALTs with preference for C6-10 or C12-14 acyl-ACP substrates. We
performed domain-swapping experiments to determine whether these features influence ALT substrate
specificity. Profiling the products of chimeric enzymes in E. coli led to the identification of amino acid
sequence fragments that affect acyl-ACP chain length preference and enzyme activity, establishing the
first links between ALT protein sequence and substrate specificity. Information from this study could be
used to engineer recombinant ALTs with substrate specificities that suit particular industrial purposes.

Rebecca Kalinger (beckykalinger@cmail.carleton.ca)

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