Structural analysis of cyclodextrin glycosyltransferase isoforms from Paenibacillus sp. A11

Abstract

The aim of this work was to determine the structure of cyclodextrin glycosyltransferase (CGTase) isoforms from Paenibacillus sp. A11, the information obtained will be used for analysis of the formation of multiple forms of the enzyme. Four CGTase isoforms of Paenibacillus sp. A11 were isolated by preparative gel electrophoresis. For the effect of post-translational modification on structure and activity of CGTase isoforms, glycosylation, phosphorylation, disulfide bond formation, and modification of carboxyl residues were studied. When enzymatic deglycosylation by the enzyme PNGase F and Endo H and chemical deglycosylation by trifluoromethanesulfonic acid (TFMS) were performed, it was found that deglycosylation, if occurred, had no effect on the size and net charge of all isoforms but exerted some effect on activity of isoforms 3 and 4. When dephosphorylation by alkaline phosphatase and investigation of disulfide bond formaion by using reducing agents were performed, it was found that both mechanisms, if occurred, had no effect on isoform pattern. Modification of carboxyl side chain with a group specific reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) suggests that amidation/deamidation had an effect on net charge of all CGTase isoforms. In the second part, the experiments were performed to analyze whether multiple forms of CGTase was the same protein. When peptides from tryptic digestion were analyzed by reverse phase HPLC on a C18 column, the number and type of peptides from all isoforms were not significantly different. Amino acid composition of all isoforms was also similar. When protein constituent in each isoform was determined by reverse phase HPLC on a C4 column, it was found that all isoforms consisted of two major forms of protein, those having Rt 10.924-10.954 and 11.951-11.595 min). When N-terminus sequence was analyzed, the first five residues of all isoforms obtained were APDTS which were the same as those of unfractionated enzyme. The result from two dimensional PAGE supports that all isoforms were rooted from a single protein. In addition, comparison of CGTase from E. coli harbouring CGTase gene of Paenibacillus sp. A11 with enzyme from original strain indicated that isoform pattern was very similar. The overall data from this work suggests that multiple forms of CGTase was the result of post-translational modification of the transcribed and translated form of the enzyme. When kinetic parameters of isoform 1 were determined, alpha-CD was the best substrate for coupling reaction (kcat/Km 3368 +- 300 min-1.mM-1) and maltohexaose was the best substrate for cyclization reaction (kcat/Km 107 +- 5 min-1.mM-1).