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D302A
almost complete loss of activity
D302A/D316A
very low residual activity
D302C
kcat value is 9.5% that of wild-type GTB
D302E
kcat value is 47% that of wild-type GTB
D302E/D316E
almost complete loss of activity
D302L
almost complete loss of activity
D302N
almost complete loss of activity
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
G176R/P234S/S235G/M266L/A268G
expressed in Escherichia coli BL21, although 4 of the mutations correspond to a change from A- to B-blood group, the P234S-mutation shows a remarkable increase in A-donor specificity
R188K
almost complete loss of activity
additional information
construction of GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S
additional information
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generation of a chimeric histo-blood group B transferase by replacing the N-acetyl-D-galactosamine recognition domain of human A transferase with the galactose-recognition domain of evolutionarily related murine alpha1,3-galactosyltransferase, the chimeric mutant shows a functional conversion from A to B transferase activity, overview. The AT-MluI-GT construct, which expresses the chimera between the long N-terminal sequence up to the MluI site of human A transferase, and the short C-terminal sequence up to the MluI site of murine a1-3Gal transferase, produces the antigens that react with both the anti-A and the Griffonia simplicifolia Lectin I, GSL-IA4, lectin
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
expression of constructs, having either of AlaGlyGly, GlyGlyAla, HisAlaAla, LeuGlyGly, or MetGlyAla tripeptide sequence at codons corresponding to 266-268 of human GTA/GTB. The original human GTA construct with LeuGlyGly and the substitution construct with AlaGlyGly exhibit strong GTA. Human GTA constructs with GlyGlyAla, HisAlaAla, LeuGlyGly, or MetGlyAla exhibit GTA, GTA/GTB, GTB, GTA, or GTB activity, respectively
additional information
preparation of human GTA derivative constructs containing any of the 20 amino acids at codon 69 with and without a GlyGlyAla substitution of the LeuGlyGly tripeptide at codons 266-268. In presence of the Forssman glycolipid synthase substrate, all substitution constructs at codon 69 exhibit Forssman glycolipid synthase activity. The combination with tripeptide GlyGlyAla substitution significantly increases the activity. With increased globoside availability, the native human GTA with a methionine residue at codon 69 also synthesizes Forssman antigen
additional information
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preparation of human GTA derivative constructs containing any of the 20 amino acids at codon 69 with and without a GlyGlyAla substitution of the LeuGlyGly tripeptide at codons 266-268. In presence of the Forssman glycolipid synthase substrate, all substitution constructs at codon 69 exhibit Forssman glycolipid synthase activity. The combination with tripeptide GlyGlyAla substitution significantly increases the activity. With increased globoside availability, the native human GTA with a methionine residue at codon 69 also synthesizes Forssman antigen
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