Characterization of a Class II α-mannosidase from Trichosanthes anguina seeds: Partial amino acid sequencing, secondary structure determination, and thermal/chemical unfolding.

Purification and partial biochemical characterization of snake gourd α-mannosidase (SGM) present in the seeds of Trichosanthes anguina was reported by us earlier. In the present study, we report partial amino acid sequence of this enzyme, determined by proteolytic digestion and mass spectrometric analysis, which revealed high sequence similarities to the Cucumis sativus α-mannosidase. Furthermore, finer comparisons with the sequences of α-mannosidases from the legume jack bean, and cucurbit Cucumis sativus clearly demonstrated unique structural features that are conserved among legume and non-legume mannosidases. These observations strongly suggest that SGM belongs to the family of glycoside hydrolase GH-38 Class II α-mannosidases. Extensive biophysical studies employing circular dichroism spectroscopy, differential scanning calorimetry, and fluorescence spectroscopy were carried out on SGM to characterize its secondary structure, and thermal as well as chemical unfolding. Analysis of far UV-CD spectra of SGM indicated that it contains of about 3.5% α-helices, 37.5% β-sheets, 12.1% β-turns and 46.8% unordered structures and that binding of both mannose (substrate) and swainsonine (inhibitor) have only a marginal effect on the secondary structure. Differential scanning calorimetry (DSC) revealed that thermal unfolding of the protein involves two transitions centred at ~80.8 and ~ 85 °C, which increased by ~1° and 3°, respectively, in the presence of mannose or swainsonine. Results of temperature-dependent CD spectroscopic studies are in good agreement with these results and yielded unfolding temperatures in the 81-86 °C range. Fluorescence spectroscopic studies on the chemical denaturation by gaunidinium chloride suggested that the protein undergoes a two-state transition without involving intermediates.