The enzyme, characterized from the archaeon Sulfolobus tokodaii, is also active toward alpha-D-glucosamine 1-phosphate (cf. EC 2.3.1.157, glucosamine-1-phosphate N-acetyltransferase). In addition, that enzyme contains a second domain that catalyses the activities of EC 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase, EC 2.7.7.24, glucose-1-phosphate thymidylyltransferase, and EC 2.7.7.83, UDP-N-acetylgalactosamine diphosphorylase.
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The expected taxonomic range for this enzyme is: Bacteria, Archaea
The enzyme, characterized from the archaeon Sulfolobus tokodaii, is also active toward alpha-D-glucosamine 1-phosphate (cf. EC 2.3.1.157, glucosamine-1-phosphate N-acetyltransferase). In addition, that enzyme contains a second domain that catalyses the activities of EC 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase, EC 2.7.7.24, glucose-1-phosphate thymidylyltransferase, and EC 2.7.7.83, UDP-N-acetylgalactosamine diphosphorylase.
because the multifunctional ST0452 protein is capable of catalyzing the last two reactions (Ec 2.3.1.157 and EC 2.7.7.23 (UDP-N-acetylglucosamine diphosphorylase)) of the bacteria-type four-step biosynthesis pathway of UDP-alpha-D-glucosamine from fructose 6-phosphate, the ST0452 protein plays an important role for the bacteria-type UDP-alpha-D-glucosamine biosynthesis pathway in this archaeon
because the multifunctional ST0452 protein is capable of catalyzing the last two reactions (Ec 2.3.1.157 and EC 2.7.7.23 (UDP-N-acetylglucosamine diphosphorylase)) of the bacteria-type four-step biosynthesis pathway of UDP-alpha-D-glucosamine from fructose 6-phosphate, the ST0452 protein plays an important role for the bacteria-type UDP-alpha-D-glucosamine biosynthesis pathway in this archaeon
the mutant enzyme shows 6.5times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues function cooperatively to increase N-acetylglucosamine-1-phosphate uridyltransferase activity
C-terminal deletion mutants DC005 and DC011 (deletion of the C-terminal 5 or 11 residues of the ST0452 protein) shows 20% and 38% less galactosamine-1-phosphate acetyltransferase activity than the wild-type ST0452 protein. The mutant enzyme DC011 (deletion of the C-terminal 11 residues of the ST0452 protein) shows little thermal stability at 80°C. The C-terminal domain of the ST0452 protein, with its LbetaH structure, appears to be essential for the formation of its trimeric form and, in turn, the high stability of the entire ST0452 protein. The deletion mutant enzymes DC021, DC031, DC041, DC071 and DC121, are produced in an insoluble form or aggregated immediately after purification. Mutant enzymes DC051 and DC171 can be expressed in a soluble form. Mutant enzyme DC051 becomes completely insoluble after 5 min treatment at 60°C, while mutant enzyme DC171 is insoluble after 5 min treatment at 70 °C
the mutant enzyme exhibits over 4 times higher N-acetylglucosamine-1-phosphate uridyltransferase activity, compared with that of the wild-type ST0452 protein. The three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein shows that positions 146 (Glu) and 80 (Thr) form interactions with GlcNAc, and an engineering strategy is applied to these residues to increase activity
the mutant enzyme exhibits over 4 times higher N-acetylglucosamine-1-phosphate uridyltransferase activity, compared with that of the wild-type ST0452 protein. The three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein shows that positions 146 (Glu) and 80 (Thr) form interactions with GlcNAc, and an engineering strategy is applied to these residues to increase activity
mutant enzyme DC005 shows the same thermostability as wild-type ST0452 protein, whereas mutant enzyme DC011 denatures and becomes insoluble form by 5-min treatment at 80 °C. The C-terminal domain of the ST0452 protein, with its LbetaH structure, appears to be essential for the formation of its trimeric form and, in turn, the high stability of the entire ST0452 protein
Honda, Y.; Nakano, S.; Ito, S.; Dadashipour, M.; Zhang, Z.; Kawarabayasi, Y.
Improvement of ST0452 N-acetylglucosamine-1-phosphate uridyltransferase activity by the cooperative effect of two single mutations identified through structure-based protein engineering
Dadashipour, M.; Iwamoto, M.; Hossain, M.M.; Akutsu, J.I.; Zhang, Z.; Kawarabayasi, Y.
Identification of a direct biosynthetic pathway for UDP-N-acetylgalactosamine from glucosamine-6-phosphate in thermophilic crenarchaeon Sulfolobus tokodaii