Isolation and functional characterization of two thioredoxin h isoforms from grape

Area of Science:

• Plant biochemistry
• Molecular biology
• Protein science

Background:

• Thioredoxins (Trxs) are crucial for cellular processes via dithiol-disulfide exchange.
• Plants exhibit diverse Trx types distinct from animals and prokaryotes.
• Understanding plant Trx isoforms is vital for comprehending their specific roles.

Purpose of the Study:

• To isolate and characterize two novel thioredoxin h isoforms from grape (Vitis vinifera).
• To investigate the enzymatic activity and redox-dependent structural changes of VvTrx h2 and VvTrx h3.
• To assess the stability and functionality of these isoforms under thermal stress.

Main Methods:

• Isolation and cloning of VvTrx h2 and VvTrx h3 cDNAs using RACE.
• Heterologous expression of recombinant proteins in Escherichia coli.
• Enzymatic activity assays (DTT-insulin reduction, DTNB reduction) and heat treatment experiments.

Main Results:

• Both VvTrx h2 and VvTrx h3 were successfully expressed and exhibited NADPH-dependent reduction by E. coli thioredoxin reductase.
• Heat shock induced oligomerization of VvTrx h isoforms above 50°C, decreasing their activity.
• Redox-dependent structural changes to monomers significantly increased disulfide reductase activity, with notable heat stability up to 99°C.

Conclusions:

• VvTrx h2 and VvTrx h3 are functional thioredoxin h isoforms from grape berries.
• These isoforms display unique redox-dependent structural dynamics influencing their enzymatic activity.
• The proteins exhibit remarkable thermal stability, retaining activity after prolonged high-temperature exposure.