Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects.
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ABSTRACT:
SUBMITTER: Taranto F
PROVIDER: S-EPMC5343912 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polyphenol oxidases (PPOs) contain the structurally similar enzymes tyrosinases (TYRs) and catechol oxidases (COs). Two cDNAs encoding pro-PPOs from tomato (Solanum lycopersicum) were cloned and heterologously expressed in Escherichia coli. The two pro-PPOs (SlPPO1-2) differ remarkably in their activity as SlPPO1 reacts with the monophenols tyramine (kcat?=?7.94?s-1) and phloretin (kcat?=?2.42?s-1) and was thus characterized as TYR, whereas SlPPO2 accepts only diphenolic substrates like dopamine (kcat?=?1.99?s-1) and caffeic acid (kcat?=?20.33?s-1) rendering this enzyme a CO. This study, for the first time, characterizes a plant TYR and CO originating from the same organism. Moreover, X-ray structure analysis of the latent holo- and apo-SlPPO1 (PDB: 6HQI and 6HQJ) reveals an unprecedented high flexibility of the gatekeeper residue phenylalanine (Phe270). Docking studies showed that depending on its orientation the gatekeeper residue could either stabilize and correctly position incoming substrates or hinder their entrance into the active site. Furthermore, phloretin, a substrate of SIPPO1 (Km?=?0.11?mM), is able to approach the active centre of SlPPO1 with both phenolic rings. Kinetic and structural results indicate that phloretin could act as a natural substrate and connote the participation of PPOs in flavonoid-biosynthesis.
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Project description:Biochemical and structural characterization of tomato PPOs provide novel insights into substrate preference and the role of the gatekeeper residue
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Project description:Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting that PPO may have a role in the development of plant water stress and potential for photoinhibition and photooxidative damage that may be unrelated to any effects on the Mehler reaction. These results substantiate the defensive role of PPO and suggest that manipulation of PPO activity in specific tissues has the potential to provide broad-spectrum resistance simultaneously to both disease and insect pests, however, effects of PPO on postharvest quality as well as water stress physiology should also be considered. In addition to the functional analysis of tomato PPO, the application of antisense/sense technology to decipher the functions of PPO in other plant species as well as for commercial uses are discussed.
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Project description:Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale). The system comprised the Escherichia coli Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26 °C). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v(max) of 0.48 ± 0.1 µkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes.
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2017-02-09 | GSE67735 | GEO