STRUCTURE AND FUNCTION RELATIONSHIP OF AMYLOMALTASE FROM Corynebacterium glutamicum

Abstract

Amylomaltase (EC 2.4.1.25) catalyzes intramolecular and intermolecular transglucosylation reactions of glucan to yield large-ring cyclodextrin (LR-CD) and linear oligosaccharide products, respectively. The aim of this work is to investigate structure and function relationship of amylomaltase from Corynebacterium glutamicum (CgAM). Three target amino acid residues of CgAM, Y418 (at the tip of 410s loop which lies over the active site), H461 (in active site) and N287 (in second glucan binding site) were selected for site-directed mutagenesis. Biochemical characterization of all purified mutated CgAMs were compared to wild-type (WT). All enzymes had an approximate size of 84 kDa on SDS-PAGE, the same optimum temperature and pH of 40 °C, pH 6.0 and 30 °C, pH 6.0 for disproportionation and cyclization reactions, respectively. For substrate specificity in disproportination reaction of WT, Y418 and N287 mutated CgAMs preferred maltotriose while maltose was suitable for H461 mutants. Significant decrease in transglucosylation activities of mutated CgAMs was observed especially cyclization activity which was lost in H461 mutants. The catalytic efficiency, kcat/Km values of all mutated CgAMs were lower than that of WT, mainly due to the significant decrease in kcat for disproportionation and cyclization reactions. Km values of Y418 mutants were not changed much while values of H461 and N287 mutants were higher than WT. In addition, Km values of Y418 and N287 mutants were higher than WT with pea starch substrate in cyclization reaction. The changes in size and yield of LR-CD products were observed as the result of Y418 and N287 mutations. Y418A/S/D mutated CgAMs gave a larger CD size (CD36 - CD40), Y418R/W showed a broad product pattern while N287A/S/D/R/W gave a smaller size (CD26 - CD28), all mutated enzymes showed lower product yield in comparison to WT with CD29-CD33 as main products. The secondary structure of all enzymes as analyzed was not significantly changed upon mutations. Biophysical properties of WT and Y418A/W CgAMs were determined by ITC and DSC techniques. Heat production of WT was higher than those of Y418A/W in disproportionation reaction. The product inhibition was occurred as a result of increase in maltotriose concentration. The heat capacity profile from DSC showed that peak temperatures (Tp) of WT and Y418A were similar while a shift of Tp of Y418W towards 4 °C lower was observed. The results indicated that WT and Y418A showed higher stability than Y418W CgAM. The model structures of mutated CgAMs were constructed using WT X-ray structure as template in order to help explain change in properties upon mutation. The overall results suggested that all three residues of CgAM are important for enzyme activity to a different extent. Y418 has to retain hydrophobic interactions and the suitable distance of loop channel for substrate entering, N287 should remain positively charge side chain. Both positions are also involved in controlling the amount and size of LR-CD products. While H461 is essential for enzyme catalysis especially in cyclization reaction.