The enzyme from plants and animals also has activity toward N-acetyl-alpha-D-glucosamine 1-phosphate (cf. EC 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase) [1,2].
alternatively spliced isoform to AGX1, differs from those referenced in the literature by two changes: S445G and Q454S (most probably a gene polymorphism)
The enzyme from plants and animals also has activity toward N-acetyl-alpha-D-glucosamine 1-phosphate (cf. EC 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase) [1,2].
the enzyme has multiple sugar-1-phosphate nucleotidylyltransferase, EC 2.7.7.37, and amino-sugar-1-phosphate acetyltransferase, EC 2.3.1.157, activities, overview. In addition to glucosamine-1-phosphate acetyltransferase activity, it possesses unique galactosamine-1-phosphate acetyltransferase activity. Also, the enzyme possesses GlcNAc-1-phosphate nucleotidylyltransferase, EC 2.7.7.23, and N-acetyl-D-galactosamine-1-phosphate uridyltransferase, EC 2.7.7.83, activities, as well as the expected glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24, activity
the three-dimensional structure of the ST0452 mutant Y97N is not changed by due to lack of metals but the interactions with the substrate is slightly modified, which might cause the activity to increase
the human UAP1 gene encodes two different isoforms, named AGX1 and AGX2, with AGX1 being more abundant in testis and AGX2 in somatic tissues. AGX-1 is an UDP-N-acetylgalactosamine diphosphorylase, EC 2.7.7.83, and AGX-2 is an UDP-N-acetylglucosamine diphosphorylase, EC 2.7.7.23
the naturally occuring UAP1 A229T mutation is potentially pathogenic. The A229T mutation induces structural changes, leading to reduced thermal stability and activity of the mutant compared to wild-type
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-GlcNAc from fructose 6-phosphate, the ST0452 protein plays an important role for the bacteria-type UDP-GlcNAc 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-GlcNAc from fructose 6-phosphate, the ST0452 protein plays an important role for the bacteria-type UDP-GlcNAc biosynthesis pathway in this archaeon
analysis of the overall structure of wild-type ST0452 protein (PDB ID 2GGO), residue 97 (Asn) interacts with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. UTP forms hydrogen bond interactions with seven residues, i.e. the main chain atoms of the position 8 Ala, position 9 Gly, position 12 Glu, position 79 Gly, and position 98 Gly residues and the side chain atoms of the position 13 Arg and position 73 Gln residues. The position 13 Arg and position 73 Gln residues appear to form more stable interactions than the other residues, with the position 13 Arg residue forming two hydrogen bonds with the phosphoryl group at the gamma-site and the amide group of the position 73 Gln residue forming a salt bridge with the uracil nucleobase in UTP
analysis of the overall structure of wild-type ST0452 protein (PDB ID 2GGO), residue 97 (Asn) interacts with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. UTP forms hydrogen bond interactions with seven residues, i.e. the main chain atoms of the position 8 Ala, position 9 Gly, position 12 Glu, position 79 Gly, and position 98 Gly residues and the side chain atoms of the position 13 Arg and position 73 Gln residues. The position 13 Arg and position 73 Gln residues appear to form more stable interactions than the other residues, with the position 13 Arg residue forming two hydrogen bonds with the phosphoryl group at the gamma-site and the amide group of the position 73 Gln residue forming a salt bridge with the uracil nucleobase in UTP
analysis of the overall structure of wild-type ST0452 protein (PDB ID 2GGO), residue 97 (Asn) interacts with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. UTP forms hydrogen bond interactions with seven residues, i.e. the main chain atoms of the position 8 Ala, position 9 Gly, position 12 Glu, position 79 Gly, and position 98 Gly residues and the side chain atoms of the position 13 Arg and position 73 Gln residues. The position 13 Arg and position 73 Gln residues appear to form more stable interactions than the other residues, with the position 13 Arg residue forming two hydrogen bonds with the phosphoryl group at the gamma-site and the amide group of the position 73 Gln residue forming a salt bridge with the uracil nucleobase in UTP
analysis of the overall structure of wild-type ST0452 protein (PDB ID 2GGO), residue 97 (Asn) interacts with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. UTP forms hydrogen bond interactions with seven residues, i.e. the main chain atoms of the position 8 Ala, position 9 Gly, position 12 Glu, position 79 Gly, and position 98 Gly residues and the side chain atoms of the position 13 Arg and position 73 Gln residues. The position 13 Arg and position 73 Gln residues appear to form more stable interactions than the other residues, with the position 13 Arg residue forming two hydrogen bonds with the phosphoryl group at the gamma-site and the amide group of the position 73 Gln residue forming a salt bridge with the uracil nucleobase in UTP
analysis of the overall structure of wild-type ST0452 protein (PDB ID 2GGO), residue 97 (Asn) interacts with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. UTP forms hydrogen bond interactions with seven residues, i.e. the main chain atoms of the position 8 Ala, position 9 Gly, position 12 Glu, position 79 Gly, and position 98 Gly residues and the side chain atoms of the position 13 Arg and position 73 Gln residues. The position 13 Arg and position 73 Gln residues appear to form more stable interactions than the other residues, with the position 13 Arg residue forming two hydrogen bonds with the phosphoryl group at the gamma-site and the amide group of the position 73 Gln residue forming a salt bridge with the uracil nucleobase in UTP
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 enzyme contains the the N-terminal nucleotidylyltransferase domain (residues 1210) and the C-terminal acetyltransferase domain (residues 211401), respectively. Comparisons of the crystal structures of the ST0452 protein, PDB ID GGO, and Escherichia coli protein EcGlmU2, PDB ID 2OI5, comparison with ST0452 mutant enzymes, overview. Despite the structural similarities between the N- and C-termini of the ST0452 protein and those of Escherichia coli EcGlmU, the thermostabilities of the two proteins differ greatly, as EcGlmU is a mesophilic enzyme. The structures of these proteins do not correlate directly with their thermostability
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant detagged AGX1A229T in complex with UDP-GlcNAc, X-ray diffraction structure determination and analysis at 1.7 A resolution, molecular replacement using the published AGX1 structure (PDB ID 1JV1) as a search model
purified recombinant enzyme mutant Y97N in complex with UTP or N-acetyl-alpha-D-glucosamine 1-phosphate, sitting drop vapor diffusion method, mixing of 11 mg/ml protein with ligands 5 mM UDP-GlcNAc and 10 mM (NH4)2SO4 (to form the complex with UDP-GlcNAc) or 5 mM UTP and 10 mM (NH4)2SO4 (to form the complex with UTP), and with cyrstallization solution containing 20% w/v PEG 3350 and 0.2 M potassium citrate tribasic monohydrate, X-ray diffraction structure determination and analysis at 2.91 and 2.09 A resolution, respectively
naturally occuring enzyme AGX1 mutation, and site-directed mutagenesis, the A229T mutation causes a reduction of protein thermal stability compared to wild-type AGX1, and AGX1A229T has lower activity in producing UDP-GlcNA. In diploid organisms, haploinsufficiency is a phenomenon in which a single copy of a functional gene is not sufficient to produce the normal/wild-type phenotype. The patient is only heterozygous for the UAP1 A229T missense mutation. The UAP1 gene is potentially haploinsufficient and LoF intolerant, and the heterozygous UAP1 A229T mutation is potentially pathogenic. The recombinant mutant enzyme shows a reduction of the melting temperature (Tm) by approximately 5.3°C compared to wild-type. The A229T mutation induces structural changes. The R228-E44 interaction is abolished in the AGX1A229T structure caused by the position shift of R228. The pushing effect is likely due to the bulkier side chain of threonine compared to that of alanine. Along with the conformational change of the N-terminal domain in the AGX1A229T structure, M218 is shifted by 0.8 A away from R169, weakening the Q112-R169-M218 interaction
site-directed mutagenesis, the mutant shows 6.5times higher activity with N-acetylglucosamine-1-phosphate compared to that of the wild-type ST0452 protein
naturally occuring mutation, the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibits over 4times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase, EC 2.7.7.23) activity, compared with that of the wild-type ST0452 protein, three-dimensional structure analysis of the Y97N protein. The overall structure is almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions
construction of expression vectors encoding a series of ST0452 C-terminal deletion mutants with hexahistidine tags at their C-termini, designated pST0452(DC005)H, pST0452(DC011)H, pST0452(DC021)H, pST0452(DC031)H, pST0452(DC041) H, pST0452(DC051)H, pST0452(DC071)H, pST0452 (DC121)H and pST0452(DC171)H. The deletion mutants retain the same tertiary structures as the wild-type ST0452 protein
all proteins substituted at position 146 have drastically decreased activities, whereas several proteins substituted at position 80 show higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein
recombinant His-tagged wild-type enzyme and mutant Y97N from Escherichia coli strain BL21-CodonPlus(DE3)-RIPL by nickel affinity chromatography and ultrafiltration
recombinant N-terminally GST-tagged wild-type and mutant AGX1 from Escherichia coli strain (DE3) pLysS by glutathione affinity chromatography, the tag is cleaved off by PreScission protease, followed by gel filtration, and ultrafiltration
gene UAP1 encodes two different isoforms, named AGX1 and AGX2, recombinant expression of N-terminally GST-tagged wild-type and mutant AGX1 in Escherichia coli strain (DE3) pLysS
Peneff, C.; Ferrari, P.; Charrier, V.; Taburet, Y.; Monnier, C.; Zamboni, V.; Winter, J.; Harnois, M.; Fassy, F.; Bourne, Y.
Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: Role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture
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
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