Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase.

Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the JCM12802 endo-polygalacturonase PGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 °C. Distinctive features were revealed by comparison of thermophilic PGA and all known structures of fungal endo-polygalacturonases, including a relatively large exposed polar accessible surface area in thermophilic PGA. By mutating potentially important residues in thermophilic PGA, we identified Thr284 as a critical residue. Mutant T284A was comparable to thermophilic PGA in melting temperature but exhibited a significantly lower half-life and half-inactivation temperature, implicating residue Thr284 in the kinetic stability of thermophilic PGA. Structure analysis of thermophilic PGA and mutant T284A revealed that a carbon-oxygen hydrogen bond between the hydroxyl group of Thr284 and the Cα atom of Gln255, and the stable conformation adopted by Gln255, contribute to its kinetic stability. Our results clarify the mechanism underlying the kinetic stability of GH28 endo-polygalacturonases and may guide the engineering of thermostable enzymes for industrial applications.