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C209A
site-directed mutagenesis, mutant structure determination
C80S/C196S
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both the double and triple mutants show differing levels of disorder depending on their liganded state, with the level of disorder increasing when substrates are bound.The double mutant shows disorder over residues 177-180 in the unliganded and H-antigen bound forms, with disorder increasing to residues 176-185 for the UDP-, UDP + H-, and UDP-alpha-D-galactose + 3-deoxy-Gal inactive acceptor analog-bound structures. The double mutant has a reduction in Km for both donor and acceptor substrates. Mutation shows little effect over kcat
C80S/C196S/C209S
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both the double and triple mutants show differing levels of disorder depending on their liganded state, with the level of disorder increasing when substrates are bound.The unliganded triple mutant shows a nearly complete internal loop, with residues 176-181 disordered for the H-antigen-bound structure and 177-179 disordered for the UDP-bound structure. The UDP + H- and UDPGal + 3-deoxy-Gal inactive acceptor analog-bound structures of the triple mutant show an internal loop completely disordered over residues 176-184. The triple Cys-to-Ser mutant has an acceptor Km elevated approximately 5times over wild type, while the donor Km has doubled. Mutation shows little effect over kcat
D302A
almost complete loss of activity
D302A/D316A
strong decrease in kcat value
D302C
kcat value is 9% that of wild-type GTB
D302E
kcat value is 47% that of wild-type GTB
D302E/D316E
50% decrease in kcat value
D302L
almost complete loss of activity
DELTA68-354
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construct in which the N-terminal transmembrane domain is deleted. Deletion results in a more crystallizable protein
E303A
residue E303 plays a critical role in maintaining the stability of a strained double-turn in the active site through several hydrogen bonds
E303C
residue E303 plays a critical role in maintaining the stability of a strained double-turn in the active site through several hydrogen bonds. Mutant retains significant activity despite disrupted active site architecture
E303D
residue E303 plays a critical role in maintaining the stability of a strained double-turn in the active site through several hydrogen bonds. Mutant retains significant activity despite disrupted active site architecture
E303Q
residue E303 plays a critical role in maintaining the stability of a strained double-turn in the active site through several hydrogen bonds. Mutant maintains active site architecture but exhibits zero activity
M186V
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site-directed mutagenesis, mutation is a naturally occuring polymorphism, amino-acid substitution in the disordered loop of the enzyme causes a weak B phenotype of erythrocytes, mutant enzyme shows reduced activity compared to the wild-type enzyme
M214G
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saturation mutagenesis of GTB enzyme at M214 leads to a two-fold higher kcat for UDP-GalNAc and specific activity of the mutant compared to the wild-type GTB
M214R
mutation is adjacent to the 211DVD213 motif. 1200fold decrease in kcat compared with wild type enzyme. The crystal structure of M214R shows that DVD motif coordination to Mn2+ is disrupted by Arg214 causing displacement of the metal by a water molecule. Individuals with the M214R mutation show the Bel variant expressing very low levels of B antigens
M214S
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saturation mutagenesis of GTB enzyme at M214 leads to a two-fold higher kcat for UDP-GalNAc and specific activity of the mutant compared to the wild-type GTB
M214T
mutation is adjacent to the 211DVD213 motif. The crystal structure of the M214T mutant shows no change in DVD motif coordination to Mn2+. Instead a critical residue, Met266, which is responsible for determining donor specificity, has adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity. Individuals with M214T mutation give rise to AweakB phenotype
M214V
mutation is adjacent to the 211DVD213 motif. Individuals with M214T mutation give rise to AweakB phenotype
P234S
dramatic and complete reversal of donor specificity, it preferentially utilizes UDP-GalNac for transfer
R188H
site-directed mutagenesis, the mutant shows affected substrate binding
S185C
mutant enzyme exhibits 4.8fold elevations in kcat/Km for UDP-glucose relative to that of wild-type enzyme
S185D
activity with UDP-galactose is 0.09% of wild-type activity
S185E
activity with UDP-galactose is 0.04% of wild-type activity
S185N
mutant enzyme exhibits 4.3fold elevations in kcat/Km for UDP-glucose relative to that of wild-type enzyme
A268T
naturally occuring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
A268T
naturally occurring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
D262N
naturally occuring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
D262N
naturally occurring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
F216I
naturally occuring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
F216I
naturally occurring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
I192T
naturally occuring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
I192T
naturally occurring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
M189V
naturally occuring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
M189V
naturally occurring mutant from individuals with weak B phenotype showing serologically weak B antigen on their red cells, overview
R188K
almost complete loss of activity
R188K
site-directed mutagenesis, the mutant shows affected substrate binding
R188S
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site-directed mutagenesis, nearly inactive mutant
R188S
site-directed mutagenesis, the mutant shows affected substrate binding
additional information
construction of GTA/GTB chimeric enzyme mutants GTB/G176R and GTB/G176R/G235S, structures of the mutants bound to a panel of donor and acceptor analogue substrates, showing open, semi-closed, and closed conformations as the enzymes go from the unliganded to the liganded states, overview
additional information
identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
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identification of naturally occruing polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
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identification of naturally occurring polymorphisms in the ABO gene, phenotypes, overview
additional information
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screening of a saturation mutagenesis library for mutant determination with altered cosubstrate specificity
additional information
ABO glycosyltransferase polymorphisms leading to reduced activity and substrate recognition, phenotyping and genotyping, overview
additional information
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construction of truncated GTB monomers composed of the full C-terminal and catalytic domain as well as a truncated N-terminal domain
additional information
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generation of a structural chemira of histo-blood group B transferase, a B type transferase, by replacing the N-acetyl-D-galactosamine recognition domain of human type A transferase with the galactose-recognition domain of evolutionarily related murine alpha1,3-galactosyltransferase, leading to functional conversion from human A to B transferase activity, overview. When the glycine 268 of the A transferase is substituted by the alanine of the B transferase, the construct expresses an enzyme with weak B transferase activity, but when the alanine 268 of B transferase is substituted by the glycine of A transferase, the construct expresses an enzyme with strong A and B transferase activity
additional information
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chimeric GTA and GTB enzymes are generated (AABB, ABBA and ABBB)
additional information
amino acids at codons 266 and 268 of human isoforms GTA, EC 2.4.1.40, and GTB, EC 2.4.1.37, are crucial to their distinct sugar specificities. In vitro mutagenized GTAs/GTBs having any of 20 possible amino acids at those codons show that those codons determine the transferase activity and sugar specificity
additional information
the enzymes GTA, EC 2.4.1.40, and GTB have nearly identical sequences, while the corresponding mutants of GTA/GTB have up to a 13fold difference in their residual activities relative to wild type.The mutated Cys, Asp and Gln functional groups are no more than 0.8 A further from the anomeric carbon of donor substrate compared to wild type