Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae

Vijayakumar Poondla, Rajasekhar Chikati, Monika Kallubai, Vidyasagar Chennupati, S. Rajagopal, Vijaya Sarathi Reddy Obulam

Research output: Contribution to journalArticle

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 Km and Vmax 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.

Original languageEnglish
Article number285
Journal3 Biotech
Volume7
Issue number5
DOIs
Publication statusPublished - Oct 1 2017

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Polygalacturonase
polygalacturonase
enzyme activity
Saccharomyces cerevisiae
Protein Isoforms
modeling
pectins
circular dichroism spectroscopy
fluorescence spectroscopy
ion
acid
metal
polysaccharide
hydrolysis
metal ions
sugar
fluorescence
Enzymes
viscosity
Circular Dichroism

Keywords

  • Circular dichroism
  • Fluorescence spectroscopy
  • Homology modeling
  • MALDI-TOF MS
  • Polygalacturonase isoforms
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Biotechnology
  • Environmental Science (miscellaneous)
  • Agricultural and Biological Sciences (miscellaneous)

Cite this

Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae. / Poondla, Vijayakumar; Chikati, Rajasekhar; Kallubai, Monika; Chennupati, Vidyasagar; Rajagopal, S.; Obulam, Vijaya Sarathi Reddy.

In: 3 Biotech, Vol. 7, No. 5, 285, 01.10.2017.

Research output: Contribution to journalArticle

Poondla, Vijayakumar ; Chikati, Rajasekhar ; Kallubai, Monika ; Chennupati, Vidyasagar ; Rajagopal, S. ; Obulam, Vijaya Sarathi Reddy. / Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae. In: 3 Biotech. 2017 ; Vol. 7, No. 5.
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AU - Chennupati, Vidyasagar

AU - Rajagopal, S.

AU - Obulam, Vijaya Sarathi Reddy

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AB - 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 Km and Vmax 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.

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