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

Thumbnail Image
Khan, Saber
Major Professor
Richard B. Honzatko
Clark F. Ford
Committee Member
Journal Title
Journal ISSN
Volume Title

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.

Thu Jan 01 00:00:00 UTC 1998