Identification of genes involved in the biosynthesis of lignans in Linum flavum
Lignans are phenolic compounds of plant secondary metabolism derived from the amino acid phenylalanine or tyrosine. Lignans are widely distributed throughout the plant kingdom and have multiple pharmaceutic-medical meanings. An example of a medically used substance is the aryltetralin lignan podophy...
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|Lignans are phenolic compounds of plant secondary metabolism derived from the amino acid phenylalanine or tyrosine. Lignans are widely distributed throughout the plant kingdom and have multiple pharmaceutic-medical meanings. An example of a medically used substance is the aryltetralin lignan podophyllotoxin (PTOX), which has cytotoxic activity. It is used to treat genital warts but is also used in the production of the PTOX derivatives etoposide and teniposide. These drugs are used to treat various types of cancer. Podophyllum spec. are currently the major source of podophyllotoxin. However, aryltetralin lignans can also be detected in some flax species (Linum spec.). The biosynthesis of aryltetralin lignans is divided into the early and late steps. While the early reaction steps have now been completely identified, some enzymes and reactions are unknown regarding the late steps. In the present work, studies on the biosynthesis of lignans such as PTOX or 6-methoxypodophyllotoxin (MPTOX) in suspension cultures of L. flavum are described. The roles of pinoresinol-lariciresinol reductase (PLR), secoisolariciresinol dehydrogenase (SDH), deoxypodophyllotoxin 6-hydroxylase (DOP6H) and deoxypodophyllotoxin 7-hydroxylase (DOP7H) are of particular interest. Enzyme DOP6H and DOP7H were already characterised in Linum spec. and potentially belong to CYP family. Hence, the identification of NADPH cytochrome P450 reductase (CPR), which is essential for cytochrome P450-dependent reactions, is also an objective in this work.
In the beginning, the RNA and gDNA were isolated from the suspension cultures of L. flavum. From RNA two CPRs were successfully identified and characterised. In order to calculate the apparent Km-values, enzyme assays were performed with varying concentrations of NADPH and cytochrome c. Both CPRs 66401 and 4753 showed high enzymatic activity towards cytochrome c with the Km-value of 8.15 µM and 15.6 µM, respectively. Towards NADPH as an electron donor, CPR 66401 showed the Km-value of 29.6 μM and CPR 4753 had the Km-value of 45.2 μM. Two binding sites each for FMN, FAD, and NADPH and one binding site for cytochrome P450 were obtained from the sequences of two CPRs. Based on the membrane anchor regions at the N-terminal CPR 66401 and CPR 4753 are classified into class I and II, respectively, according to Ro et al. (2002).
PLR is a bifunctional enzyme that catalyses the formation of secoisolariciresinol from pinoresinol via lariciresinol. From the transcriptome of L. flavum, one contig with the highest similarity with PLR was found. This contig was successfully isolated from the RNA of suspension cultures of L. flavum and subsequently characterised as PLR. PLR of L. flavum catalyses stereospecifically the conversion of (+)-pinoresinol via (+)-lariciresinol into (-)-secoisolariciresinol. PLR displayed a Km-value for NADPH of 22.4 μM and has the highest catalytic activity at pH of 7 and temperature from 27 °C to 33 °C. Furthermore, in order to investigate the role of the varying size of important amino acids in the binding pocket two mutants of PLR G280Y and Y284G were constructed. The loss of enzymatic activity of both mutants LfPLR G280Y and Y284G showed the crucial role of two positions G280 and Y284 for the catalytic activity of this enzyme.
In addition, multiple candidates of CYP and SDH were obtained in the transcriptome of L. flavum. Eleven open reading frames (ORFs) of CYP and five ORFs of SDH were successfully amplified from RNA or gDNA of L. flavum. They were also successfully expressed in E. coli and yeast. The conserved domain for CYP and SDH are observed in the sequences of each candidate. Nevertheless, no recombinant proteins showed any activity in enzyme assays. Maybe a post-translational modification is essential for these enzymes and bacteria and yeast are not sufficient to conduct the required modifications. A higher expression system, for example in plant or insect, could be a solution to this problem. The searching for candidates should be refined to look for other candidates for CYP and SDH as well.