Chapter 3 - Improvement of the Amino-Sugar-1-Phosphate Acetyltransferase Activity of the Archaeal Bifunctional Protein

Abstract

The ST0452 protein was originally detected within the genomic data of the thermophilic archaeon Sulfolobus tokodaii as glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) on the basis of sequence similarity. The ST0452 protein exhibited novel galactosamine-1-phosphate acetyltransferase (GalN-1-P AcTase) activity as well as glucosamine-1-phphate acetyltransferase (GlcN-1-P AcTase), and multiple sugar-1-phosphate nucleotidylyltransferase activities. The ST0452 N-acetylglucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity was successfully increased without decreasing its thermostability by substituted mutagenesis into amino-acid residues within the reaction center to overcome the lower activity of the wild-type ST0452 protein compared with that of the bacterial counterpart enzymes. This review gives an overview of the ST0452 protein then attempts to characterize the important residues for the amino-sugar-1-P AcTase activity coded in its C-terminal region. Substitution mutations were introduced into the reaction center and a short truncation from its C-terminus was introduced to characterize the regions responsible for its acetyltransferase activity. From a construction process that resulted in a number of mutant proteins, some number of the mutant ST0452 proteins exhibiting drastically increased GlcN-1-P AcTase activity were obtained. The strategy used to increase the GlcN-1-P AcTase activity of the ST0452 protein might provide a powerful tool for the production of an improved thermostable enzyme.