Identification of essential tryptophan in amylomaltase from Corynebacterium glutamicum.
This work aims to identify essential tryptophan residue(s) of amylomaltase from Corynebacterium glutamicum (CgAM) through chemical modification and site-directed mutagenesis techniques. The recombinant enzyme expressed by Escherichia coli was purified and treated with N-bromosuccinimide (NBS), a modifying agent for tryptophan. A significant decrease in enzyme activity was observed indicating that tryptophan is important for catalysis. Inactivation kinetics with NBS resulted in pseudo first-order rate constant (kinact) of 2.31 min(-1). Substrate protection experiment confirmed the active site localization of the NBS-modified tryptophan residue(s) in CgAM. Site-directed mutagenesis was performed on W330, W425 and W673 to localize essential tryptophan residues. Substitution by alanine resulted in the loss of intra- and intermolecular transglucosylation activities for all mutated CgAMs. Analysis of circular dichroism spectra showed no change in the secondary structure of W425A but a significant change for W330A and W673A from that of the WT. From these results in combination with X-ray structural data and interpretation from the binding interactions in the active site region, W425 was confirmed to be essential for catalytic activity of CgAM. The hydrophobicity of this tryptophan was thought to be critical for substrate binding and supporting catalytic action of the three carboxylate residues at the active site.
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Wanitcha
W
Rachadech
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Pitchanan
P
Nimpiboon
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Wachiraporn
W
Naumthong
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand.
Santhana
S
Nakapong
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Kuakarun
K
Krusong
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: piamsook.p@chula.ac.th.
Piamsook
P
Pongsawasdi
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D048230:Corynebacterium glutamicum / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*
D006004:Glycogen Debranching Enzyme System; D011994:Recombinant Proteins; D014364:Tryptophan; C020569:4 alpha-glucanotransferase
Amylomaltase; Chemical modification; Essential tryptophan; N-bromosuccinimide; Site-directed mutagenesis