Biophysical studies of catalytic and starch binding domains of wild-type and mutant glucoamylases from Aspergillus awamori

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1998
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Khan, Saber
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Richard B. Honzatko
Clark F. Ford
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Aspergillus awamori catalytic and starch binding domains were prepared, purified and characterized. Sedimentation equilibrium experiments and MALDI-TOF spectra reveals that GA470 and SBD contains ~12% and 5% carbohydrate, respectively. Temperature denaturation experiments indicate that catalytic domain unfolds irreversibly whereas that of SBD is reversible with DeltaH of ~410 and 71 kcal/mol, respectively, and T m of 60.9° and 51.3°C, respectively. Analysis of scan rate dependency data suggest that the thermal unfolding of GA470 was partially under kinetic control and do not follow a single two-step model. Comparison of Tm of isolated and intact GA470 and SBD indicates that catalytic domain/linker thermally stabilizes the binding domain by at least 5°C. Solvent accessible surface area calculation along with experimentally determined DeltaH values indicate that glycosylation effect DeltaH as much as 59 kcal/mol for GA470 and 13 kcal/mol for SBD;The three dimensional structures of thermostable glucoamylase mutants, N20C/A27C, N20C/A27C/G137A and N20C/A27C/S30P/G137A were determined at 2.5, 2.4 and 2.3 A resolution by X-ray crystallography. The engineered disulfide bond is right-handed with an average strain energy of 4 kcal/mol and unusual average dihedral angle, c'2 = 93°. Crystallographic studies show that introduced proline at position 30 adopts a trans conformation, &phis; = -64°, and psi = 131°. We did observe a decrease in the thermal factors of the main chain atoms of proline at site 30 (12 A versus 20 A); however, thermal factors in and around 20--27 loop, G137A and elsewhere in mutant proteins were found to be essentially the same to that of wild-type enzyme. Crystallographic analysis also reveals that replaced Alanine 137 was accommodated within helix-4, supporting the empirical observation that Alanine 137 has high helix forming propensity. Enzymatic thermostability appears to be correlated with the relative accessibility of the substituted residues and a decrease in backbone entropy of unfolding by reducing backbone flexibility. Although, the overall structures of the mutants are very similar compared to wild-type, there are some local conformation differences as indicated from r.m.s. deviations values calculated within 6 A from the mutation site, which supports that the effects of combined mutation in N20C/A27C/S30P/G137A are cumulative.

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Thu Jan 01 00:00:00 UTC 1998