Identification of essential tryptophan in amylomaltase from Corynebacterium glutamicum

International Journal of Biological Macromolecules
Wanitcha RachadechPiamsook Pongsawasdi

Abstract

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 activit...Continue Reading

References

Aug 31, 2013·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Wiraya SrisimaratKuakarun Krusong

❮ Previous
Next ❯

Citations

Feb 16, 2016·International Journal of Biological Macromolecules·Pitchanan NimpiboonPiamsook Pongsawasdi
May 8, 2016·Cellular and Molecular Life Sciences : CMLS·Štefan Janeček, Marek Gabriško
Jul 2, 2016·Journal of Agricultural and Food Chemistry·Seongjoon JooKyung-Jin Kim
Aug 11, 2016·Biochemical and Biophysical Research Communications·Pitchanan NimpiboonPiamsook Pongsawasdi

❮ Previous
Next ❯

Related Concepts

Related Feeds

Cardiac Glycosides

Cardiac glycosides are a diverse family of naturally derived compounds that bind to and inhibit na+/k+-atpase. Discover the latest research on cardiac glycosides heres.