02139nas a2200217 4500008004100000022001400041245012400055210006900179260007500248300001200323490000700335520131900342653002201661653001701683653001701700653001301717653002101730100002401751700002501775856012101800 2007 eng d a1053-050900aFluorescence quenching and time-resolved fluorescence studies of alpha-Mannosidase from Aspergillus fischeri (NCIM 508)0 aFluorescence quenching and timeresolved fluorescence studies of  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScNOV a599-6050 v173 a

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.

10aalpha-Mannosidase10aDenaturation10afluorescence10alifetime10aSolute quenching1 aShashidhara, K., S.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/fluorescence-quenching-and-time-resolved-fluorescence-studies-alpha-mannosidase-001544nas a2200205 4500008004100000022001400041245009700055210006900152260007300221300001200294490000700306520077500313653002201088653002501110653001301135653001601148100002401164700002501188856012501213 2009 eng d a0141-813000aClass II alpha-mannosidase from aspergillus fischeri: energetics of catalysis and inhibition0 aClass II alphamannosidase from aspergillus fischeri energetics o aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJAN a112-1150 v443 a

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.

10aalpha-Mannosidase10aEnergy of activation10akinetics10aSwainsonine1 aShashidhara, K., S.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/class-ii-alpha-mannosidase-aspergillus-fischeri-energetics-catalysis-and-inhibition-002163nas a2200253 4500008004100000022001400041245013100055210006900186260011700255300001000372490000600382520116900388653001601557653002201573653002601595653002701621653001501648100002401663700002001687700002601707700003101733700001501764856013001779 2009 eng d a0973-626300aInteraction of alpha-mannosidase from aspergillus fischeri with glycosidase inhibitors, metal ions and group specific reagents0 aInteraction of alphamannosidase from aspergillus fischeri with g aSECTOR A-80, SCHEME NO 54, VIJAY NAGAR, A B ROAD, INDORE, 452 010 MP, INDIAbRESEARCH JOURNAL BIOTECHNOLOGYcNOV a39-480 v43 a

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.

10aactive-site10aalpha-Mannosidase10aChemical modification10aGlycosidase inhibitors10ametal ions1 aShashidhara, K., S.1 aGaikwad, S., M.1 aKhan, Mohammad, Islam1 aBharadwaj, Kishor, Chandra1 aPandey, G. uhttps://library.ncl.res.in/content/interaction-alpha-mannosidase-aspergillus-fischeri-glycosidase-inhibitors-metal-ions-and-001944nas a2200253 4500008004100000022001400041245010200055210006900157260007500226300001200301490000700313520104400320653001601364653002201380653001601402653002301418653001701441653001101458653001601469653002501485100002401510700002501534856013101559 2010 eng d a1053-050900aConformational and functional transitions in class II alpha-mannosidase from aspergillus fischeri0 aConformational and functional transitions in class II alphamanno a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScJUL a827-8360 v203 a

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.

10aAggregation10aalpha-Mannosidase10aANS binding10aCircular dichroism10afluorescence10aGdnHCl10aHydrobicity10aThermal denaturation1 aShashidhara, K., S.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/conformational-and-functional-transitions-class-ii-alpha-mannosidase-aspergillus-fischeri-0