Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae

Vijayakumar Poondla; Rajasekhar Chikati; Monika Kallubai; Vidyasagar Chennupati; Rajagopal Subramanyam; Vijaya Sarathi Reddy Obulam

doi:10.1007/s13205-017-0912-5

Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae

3 Biotech. 2017 Oct;7(5):285. doi: 10.1007/s13205-017-0912-5. Epub 2017 Aug 17.

Authors

Vijayakumar Poondla¹, Rajasekhar Chikati², Monika Kallubai³, Vidyasagar Chennupati¹, Rajagopal Subramanyam³, Vijaya Sarathi Reddy Obulam¹

Affiliations

¹ Department of Biochemistry, Sri Venkateswara University, Tirupati, 517 502 India.
² Department of Biochemistry, Osmania University, Hyderabad, 500 007 India.
³ Department of Plant Sciences, University of Hyderabad, Hyderabad, 500 046 India.

Abstract

Earlier, low-temperature-active polygalacturonase isoforms from Saccharomyces cerevisiae PVK4 were isolated and purified. Substrate specificity of polygalacturonase isoforms indicated high affinity for pectins and very low enzyme activity towards non-pectic polysaccharides. To characterize the polygalacturonase isoforms, biochemical, spectral, and in silico approaches were used. The apparent K_m and V_max values for hydrolysis of pectin and galacturonic acid were 0.31 mg/ml and 3.15 mmol min/mg, respectively. Interestingly, the polygalacturonase isoforms were found to be more stable at optimal pH and temperature of 4.5 and 40 °C, respectively. These isoforms were reacted with different metal ions; Cd²⁺ and Ni²⁺ severely inhibited the enzyme activity, while Mg²⁺, Zn²⁺, Cd²⁺, Fe²⁺ Cu²⁺, and Ni²⁺ inhibited to a lesser extent, which clearly demonstrated that variations in enzyme activity were due to their differential binding affinity of metal ions. Furthermore, decrease in the viscosity of polygalacturonic acid and citrus pectin by these isoforms was approximately four and six times higher than the rate of release of reducing sugars. This indicates that polygalacturonase isoforms have an endo-mode of action. In addition to the above, thermostability of purified polygalacturonase isoforms was studied by circular dichroism and fluorescence spectroscopy. Circular dichroism showed 18% alpha helix and 57% beta sheets at pH 5, while at pH 7, 8, and 9 there was an increase of random coil. Fluorescence studies revealed small conformational changes, which were observed at 30-50 °C, while unfolding transition region was noticed between 60 and 70 °C. The purified enzyme fractions were analyzed by MALDI-TOF MS. Finally, 3D model structures for isoenzymes of polygalacturonase of S. cerevisiae were generated and validated as good quality models, which are also suitable for molecular interaction studies.

Keywords: Circular dichroism; Fluorescence spectroscopy; Homology modeling; MALDI-TOF MS; Polygalacturonase isoforms; Saccharomyces cerevisiae.