Phytochemistry p. 1564 - 1571 (2006)
Update date:2022-08-04
Topics:
Prosser, Ian M.
Adams, Racheal J.
Beale, Michael H.
Hawkins, Nathan D.
Phillips, Andrew L.
Pickett, John A.
Field, Linda M.
Using oligonucleotide primers designed to the known gene sequence of an (E)-β-farnesene (EβF) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Mentha × piperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the EβF synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a Km for FPP of 1.91 ± 0.1 μM and kcat of 0.18 s-1, and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from EβF synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in EβF synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored EβF synthase activity (Km for FPP 0.98 ± 0.12 μM, kcat 0.1 s-1), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that EβF was not present, confirming that this particular mint chemotype had lost EβF synthase activity due to the observed mutations.
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