Reinigung und Charakterisierung einer Dehydrogenase des Phloridzinbiosynthesewegs

Abstract

Phloridzin along with its derivatives belong to the dihydrochalcones, a rarely encountered subgroup of the flavonoids. The synthesis of it is limited to a few plant species, of which only Malus produces significant amounts. In Malus, phloridzin can make up 90 % of all soluble phenols. It exhibits several pharmacological effects upon digestion or injection and was the subject of thorough research regarding this since its discovery in 1835. This was mostly in regards to glucose uptake. It is also of interest for agricultural and plant-biotechnological applications. Recently, scientific research has aimed at elucidating the biosynthesis and relevance in planta of phloridzin. The formation of phloridzin was determined as a three-step enzymatic reaction diverging from p-coumaroyl-CoA, which is an important precursor for flavonoid biosynthesis. p-Coumaroyl-CoA is reduced to dihydro-p-coumaroyl-CoA by a reductase, which is then taken up by chalcone synthase (CHS) to form phloretin, followed by glycosylation to generate phloridzin. The enzyme catalysing the reduction (in this thesis addressed as p-coumaroyl-CoA reductase (PCCR)) has been identified as the probable key enzymatic step for phloridzin formation, as both chalcone synthase (CHS) and glycosyltransferases that accept phloretin are present in plant species that are not able to produce it. PCCR could not be confidently identified as of now, although there are several candidates found by genomic searches. This thesis presents the isolation and purification of an enzyme capable of PCCR activity from young apple leaves. By a combination of two Aqueous Two Phase Systems (ATPS) and protein precipitation, the soluble leaf cell proteins could be separated from interfering compounds such as phenols. With several applications of ion exchange chromatography, an enzyme with PCCR capabilities was attained in near purity. By electrophoresis purity was achieved and the resulting protein was sequenced. A gene that would transcribe the found protein was discovered in the M. × domestica genome. It was discovered that it was already known in a theoretical fashion, as a predicted trans-2-enoyl-CoA reductase, found by a gene-prediction algorithm. A subcellular location prediction programme determined the protein to be mitochondrial localized, recognizing a transit-peptide at the N-terminus. A search for known structural homologues resulted in a human protein that exhibits 50 % sequence identity. It was identified as a mitochondrial enoyl-(acyl-carrier-protein) reductase, which catalyses a similar reaction to PCCR, a thioester moiety α-β double bond reduction. Furthermore, a homologue from rat exhibits an isoform that is formed by alternative splicing, lacking a mitochondrial transit peptide and other amino-acids. Based on this, the hypothesis was formed that this could also be the case for PCCR, which would fit with the fact that most enzymes of the flavonoid biosynthesis are either membrane bound to the ER or soluble in the cytosol. For continuative studies, the coding sequence of PCCR was isolated and amplified by mRNA extraction and reverse transcription. Three length-variants of the sequences were cloned into bacterial expression vectors for heterologous production, the original length as well as one without transit peptide and one resembling an alternatively spliced isoform, derived from the mentioned rat enoyl-(acyl-carrier-protein) reductase. PCCR was expressed as a functionally active enzyme, although in too low amount for enzymatic characterization. For investigation of the physiological role of PCCR and the function of phloridzin in planta, both a silencing vector and overexpression vectors were constructed using the PCCR coding sequence. A CRISPR-Cas9 was constructed with a gRNA binding site in the PCCR locus, which’s application is likely to silence the gene. Using the GoldenBraid 2.0 system, three plant overexpression vectors were constructed, one containing each of the aforementioned length variants of PCCR. With these, the effects of a high concentration of PCCR in living plants can be studied in the future.