@article { ISI:000250755100004, title = {Fluorescence quenching and time-resolved fluorescence studies of alpha-Mannosidase from Aspergillus fischeri (NCIM 508)}, journal = {Journal of Fluorescence}, volume = {17}, number = {6}, year = {2007}, month = {NOV}, pages = {599-605}, publisher = {SPRINGER/PLENUM PUBLISHERS}, type = {Article}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

Apart from the vital role in glycoprotein biosynthesis and degradation, alpha-mannosidase is currently an important therapeutic target for the development of anticancer agents. Fluorescence quenching and time-resolved fluorescence of alpha-mannosidase, a multitryptophan protein from Aspergillus fischeri were carried out to investigate the tryptophan environment. The tryptophans were found to be differentially exposed to the solvent and were not fully accessible to the neutral quencher indicating heterogeneity in the environment. Quenching of the fluorescence by acrylamide was collisional. Surface tryptophans were found to have predominantly positively charged amino acids around them and differentially accessible to the ionic quenchers. Denaturation led to more exposure of tryptophans to the solvent and consequently in the significant increase in quenching with all the quenchers. The native enzyme showed two different lifetimes, tau (1) (1.51 ns) and tau (2) (5.99 ns). The average lifetime of the native protein (tau) (3.187 ns) was not affected much after denaturation (tau) (3.219 ns), while average lifetime of the quenched protein samples was drastically reduced (1.995 ns for acrylamide and 1.537 ns for iodide). This is an attempt towards the conformational studies of alpha-mannosidase.

}, keywords = {alpha-Mannosidase, Denaturation, fluorescence, lifetime, Solute quenching}, issn = {1053-0509}, doi = {10.1007/s10895-007-0227-8}, author = {Shashidhara, K. S. and Gaikwad, Sushama M.} } @article { ISI:000262769700020, title = {Class II alpha-mannosidase from aspergillus fischeri: energetics of catalysis and inhibition}, journal = {International Journal of Biological Macromolecules}, volume = {44}, number = {1}, year = {2009}, month = {JAN}, pages = {112-115}, publisher = {ELSEVIER SCIENCE BV}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, abstract = {

Energetics of the catalysis of Class II alpha-mannosidase (E.C.3.2.1.24) from Aspergillus fischeri was studied. The enzyme showed K(cat)/K(m) for Man (alpha 1-3) Man, Man (alpha 1-2) Man and Man (alpha 1-6) Man as 7488, 5376 and 3690 M(-1) min(-1), respectively. The activation energy, Ea was 15.14, 47.43 and 71.21 kJ/mol for a1-3, alpha 1-2 and alpha 1-6 linked mannobioses, respectively, reflecting the energy barrier in the hydrolysis of latter two substrates. The enzyme showed K(cat)/K(m) as 3.56 x 10(5) and 4.61 x 10(5) M(-1) min(-1) and E(a) as 38.7 and 8.92 kJ/mol, towards pNP alpha Man and 4-MeUmb alpha Man, respectively. Binding of Swainsonine to the enzyme is stronger than that of 1-deoxymannojirimycin. (C) 2008 Elsevier B.V. All rights reserved.

}, keywords = {alpha-Mannosidase, Energy of activation, kinetics, Swainsonine}, issn = {0141-8130}, doi = {10.1016/j.ijbiomac.2008.10.012}, author = {Shashidhara, K. S. and Gaikwad, Sushama M.} } @article { ISI:000272134300006, title = {Interaction of alpha-mannosidase from aspergillus fischeri with glycosidase inhibitors, metal ions and group specific reagents}, journal = {Research Journal of Biotechnology}, volume = {4}, number = {4}, year = {2009}, month = {NOV}, pages = {39-48}, publisher = {RESEARCH JOURNAL BIOTECHNOLOGY}, address = {SECTOR A-80, SCHEME NO 54, VIJAY NAGAR, A B ROAD, INDORE, 452 010 MP, INDIA}, abstract = {

As an initial step towards using alpha-mannosidase as a target against anticancer drugs, inhibition studies of a model enzyme, class II alpha-mannosidase from Aspergillus fischeri in presence of polyhydroxy piperidine derived glycosidase inhibitors, metal ions and amino acid specific reagents were carried out to reveal the sensitivity of the enzyme. Three of the derivatives (Compound 20, 32 and 39)(11) showed competitive inhibition (K(i) =45, 48 and 235 mu M) and the binding of the inhibitors to the enzyme was entropically driven. Among the metal ions checked, Cu(++) (K(i) = 21nm) and Se(++) ions (K(i) = 32 mu M) showed noncompetitive and Co(++) (K(i) = 1.195 mM) showed competitive inhibition of the enzyme activity with insignificant change in the secondary structure of the protein. The above studies exhibit the Potential of the enzyme in studying anticancer drugs. Treatment of the enzyme with group specific reagents showed the presence of carboxylate, Arg and Cys at the active site. Substrate protection studies and kinetics of the modified enzyme confirmed the above results. Trp and His at the active site were observed to be in proximity.

}, keywords = {active-site, alpha-Mannosidase, Chemical modification, Glycosidase inhibitors, metal ions}, issn = {0973-6263}, author = {Shashidhara, K. S. and Gaikwad, S. M. and Khan, Mohammad Islam and Bharadwaj, Kishor Chandra and Pandey, G.} } @article { ISI:000280597500004, title = {Conformational and functional transitions in class II alpha-mannosidase from aspergillus fischeri}, journal = {Journal of Fluorescence}, volume = {20}, number = {4}, year = {2010}, month = {JUL}, pages = {827-836}, publisher = {SPRINGER/PLENUM PUBLISHERS}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

The conformational transitions in an oligomeric and high molecular weight class II alpha-mannosidase from Aspergillus fischeri were examined using fluorescence and CD spectroscopy under chemical, thermal and acid denaturing conditions. The enzyme lost the activity first and then the overall folded conformation and secondary structure. The midpoint values of GdnHCl mediated changes measured by inactivation; fluorescence and negative ellipticity were 0.48 M, 1.5 M and 1.9 M, respectively. The protein almost completely unfolded in 4.0 M GdnHCl but not at 90 A degrees C. The inactivation and unfolding were irreversible. At pH 2.0, the protein exhibited molten-globule like intermediate with rearranged secondary and tertiary structures and exposed hydrophobic amino acids on the surface. This species showed increased accessibility of Trp to the quenchers and got denatured with GdnHCl in a different manner. The insoluble aggregates of a thermally denatured protein could be detected only in the presence of 0.25-0.75 M GdnHCl.

}, keywords = {Aggregation, alpha-Mannosidase, ANS binding, Circular dichroism, fluorescence, GdnHCl, Hydrobicity, Thermal denaturation}, issn = {1053-0509}, doi = {10.1007/s10895-010-0625-1}, author = {Shashidhara, K. S. and Gaikwad, Sushama M.} }