Expression of deletion constructs of bovine beta-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity.
Bovine beta-1,4-galactosyltransferase (beta-1,4-GT; EC 2.4.1.90) belongs to the glycosyltransferase family and as such shares a general topology: an N-terminal cytoplasmic tail, a signal anchor followed by a stem region and a catalytic domain at the C-terminal end of the protein. cDNA constructs of the N-terminal deleted forms of beta-1,4-GT were prepared in pGEX-2T vector and expressed in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCl and required an 'oxido-shuffling' reagent for regeneration of the enzyme activity. The recombinant beta-1,4-GT, devoid of the GST domain, has 30-85% of the sp. act. of bovine milk beta-1,4-GT with apparent Kms for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme. Deletion analyses show that both beta-1,4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-d129). The activities are lost when either deletions extend up to residue 142 (pGT-d142) or Cys134 is mutated to Ser (pGT-d129C134S). These results suggest that the formation of a disulfide bond involving Cys134 holds the protein in a conformation that is required for enzymatic activity.
Boeggeman
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EE
Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Balaji
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PV
Sethi
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N
Masibay
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AS
Qasba
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PK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D009237:N-Acetyllactosamine Synthase; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine