Heterologe Expression und physikochemische Charakterisierung der Polyphenoloxidasen aus dem Cabernet Sauvignon (Vitis vinifera)
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Abstract
Type-3 copper proteins are ubiquitously distributed. Polyphenol oxidases (PPOs), which are characteristic for green plants, are classified as enzymatic tyrosinases and catechol oxidases.
In the present work, three PPO sequences from the grape Vitis vinifera L. cv. Cabernet Sauvignon (L-VvPPOcs-1, L-VvPPOcs-2 and L-VvPPOcs-3) were obtained from cDNA by cloning and sequencing. These were defined as two different isoforms, one of which is presumably represented by two alleles (L-VvPPOcs-1 and L-VvPPOcs-2). Here, L-VvPPOcs-1 had a sequence identity of 98.8 % to that of Virador et al. (2010) published reference structure L-VvPPOg (U83274). In addition, for L-VvPPOcs-2 and L-VvPPOcs-3 a heterologous recombinant expression system in E. coli could be established. This allowed an overexpression of the latent isoenzymes with a molecular mass of about 60 kDa as native strep-tag II fusion proteins, which could be obtained by affinity chromatography with yielded purities of 90-95 %. Subsequently the biochemically enriched latent PPOs were enzymatically characterized after activation with SDS.
Using a biochemical assay in native PAGE, the recombinant enzymes were tested for both mono- and diphenolase activities. For L-VvPPOcs-2, the results showed for the first time, a recombinant plant PPO with explicit monophenolase activity. The recombinant L-VvPPOcs-3, on the other hand, also showed clear diphenolase activity, but no significant enzymatic conversion was found for the monophenols used. Thus, L-VvPPOcs-3 was designated as catechol oxidase. Adopting PCR-based site-directed mutagenesis experiments on L-VvPPOcs-3 expression constructs, thereby substituting a glycine to an asparagine, resulted in a full conversion into a tyrosinase with defined monophenolase activity. For type-3 copper proteins, these results show the dependence of the monophenolase activity on the presence of an asparagine and a conserved glutamate, which together bind and activate an also conserved water molecule. The latter most likely mediates the deprotonation of monophenolic substrates, which allows adequate coordination of the formed phenolates to the CuA binding site. The bound phenolates can then be hydroxylated in ortho-position with the corresponding catecholates. The presented results allow the controversially discussed discrimination of catechol oxidases and tyrosinases at the molecular level.