A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids

Area of Science:

• Biochemistry
• Plant Science
• Structural Biology

Background:

• Hydrogen peroxide (H2O2) is a reactive oxygen species produced during plant metabolic processes and stress responses.
• Ascorbate peroxidase (APX) is a crucial enzyme that detoxifies H2O2, protecting cells from oxidative damage.

Purpose of the Study:

• To elucidate the crystal structure of cytosolic APX from sorghum (Sorghum bicolor).
• To investigate the unique ascorbate binding mechanisms and catalytic functions of SbAPX.

Main Methods:

• X-ray crystallography to determine the structure of sorghum APX (SbAPX).
• Steady-state enzyme kinetics assays.
• Site-directed mutagenesis to probe residue function.

Main Results:

• SbAPX exhibits a unique structure with four bound ascorbate molecules at distinct sites (ɣ-heme, δ-meso, and two surface pockets).
• Multiple binding sites facilitate positive cooperativity in ascorbate oxidation and establish a proton network.
• APX catalyzes H2O2-dependent ascorbate oxidation producing a dehydroascorbic acid derivative and potentially mediates phenylpropanoid polymerization.

Conclusions:

• Sorghum APX possesses novel ascorbate binding capabilities contributing to its H2O2 scavenging efficiency.
• The enzyme's dual role in H2O2 detoxification and phenylpropanoid polymerization suggests a mechanism for stress-induced cell wall strengthening.