Identification of Novel Acetyltransferase Activity on the Thermostable Protein ST0452 from Sulfolobus tokodaii Strain 7

Abstract

and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-D-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.The nucleotide-sugar molecule is used as a substrate for the construction of the polymer structure of carbohydrates and as a starting material for the biosynthesis of modified-sugar-nucleotide conjugates; for example, TDP-glucose is the starting material for the construction of TDP-rhamnose (25). Several sugar-1-phosphate nucleotidylyltransferases (sugar-1-P NTases) involved in some biosynthetic reactions, such as glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase), N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase), and mannose-1-phosphate guanylyltransferase, have been identified from many bacteria, including pathogens, as well as from eukaryotes (5,10,11,17,25,27,29). However, only limited information on the archaeal enzyme with sugar-1-P NTase activity has been obtained.Our group has previously reported the first thermostable enzyme with sugar-1-P NTase activity from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7 (31). The sugar-1-P NTase activity of the ST0452 protein, which was primarily identified by sequence similarity with the Escherichia coli RmlA protein, indicated the utilization of Glc-1-P and GlcNAc-1-P as a sugar-1-P substrate and all deoxynucleoside triphosphates (dNTPs) and UTP as nucleoside triphosphate (NTP) substrates, but glucosamine-1-phosphate (GlcN-1-P) was not utilized as a substrate. It was also reported that the GlcNAc-1-P UTase activity of the ST0452 protein was improved by the introduction of site-directed mutagenesis (30).The identification of GlcNAc-1-P UTase activity on the ST0452 protein suggests that the protein may have activities and features similar to those of the bacterial GlmU enzyme (19). However, the ST0452 protein exhibits only a low level of sequence identity (less than 20% identity) with E. coli GlmU (data not shown). The phylogenetic analysis of the ST0452 protein and related proteins indicates that the ST0452 protein and homologous proteins form a group independent of other related enzymes identified as RmlA or GlmU (Fig. 1). In spite of the low level of sequence similarity of the ST0452 protein with the bacterial GlmU, GlcNAc-1-P UTase activity was detected on the protein. Twe...

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position

“…Our investigations to date have shown that the ST0452 protein exhibits high thermostability, Glc-1-P TTase activity as predicted, and unexpectedly high N-acetyl-Dglucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity ( Fig. 1) as well as amino-sugar-1-phosphate acetyltransferase (amino-sugar-1-P AcTase) activity (4,5). Comparison of the activities of the ST0452 protein to those of similar enzymes from bacteria showed that both the apparent K m and k cat values of the ST0452 GlcNAc-1-P UTase activity were smaller than those of Escherichia coli GlmU (EcGlmU) enzymes (6,7).…”

Reversal of heavy metal-induced antibiotic resistance by dandelion root extracts and taraxasterol

“…Bacteria-secreted β-lactamase can be activated under various stresses and provides multi-resistance against β-lactam antibiotics (such as ampicillin) by breaking the drug's structure and deactivating the antibacterial properties [20]. Similarly, aminoglycoside bactericidal antibiotics such as kanamycin can be modified by a group of acetyltransferases via acetylation, leading to drug inactivation [21]. Both enzymes have also been shown to be stimulated by heavy metals [26,36].…”

“…For analysis of acetyltransferase, 10 µg protein was mixed with 2 mM acetyl-CoA and 2 mM amino sugar in 150 µl of 50 mM Tris-HCl at 80 °C for 30 min. After that, 0.5 mM DTNB in 50 µl of 50 mM Tris-HCl was added for 5 min following detection of absorbance at 410 nm [21].…”

“…The activity of β-lactamase or acetyltransferase is often higher in drug-resistant bacteria, leading to the inactivation of antibiotics [20,21,26]. The activity of β-lactamase was significantly induced by ampicillin ( Fig.…”

Improvement of ST0452 N -Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering

“…In the thermophilic archaeon Sulfolobus tokodaii, the ST0452 protein (20) was originally detected as a glucose-1-phosphate (Glc-1-P) thymidylyltransferase (TTase) by a similarity search within the genomic data (21). Functional analyses of the recombinant ST0452 protein produced in Escherichia coli indicated that the protein exhibited both multiple sugar-1-P nucleotidylyltransferase (NTase) activities, including N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) and Glc-1-P UTase activities, and multiple amino-sugar-1-P acetyltransferase activities, including glucosamine-1-phosphate acetyltransferase and galactosamine-1-phosphate acetyltransferase activities (20,22,23).…”