Role of a conserved acidic cluster in bovine beta1,4 galactosyltransferase-1 probed by mutagenesis of a bacterially expressed recombinant enzyme

The truncated catalytic domain of bovine beta1,4 galactosyltransferase-1 was expressed as inclusion bodies in E.coli and folded to generate 10-15 mg of active enzyme per liter of bacterial culture after extraction and purification under denaturing conditions. Mutations were introduced to investigate...

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Bibliographic Details
Published in:Glycobiology (Oxford) 1999-08, Vol.9 (8), p.815-822
Main Authors: Zhang, Y, Malinovskii, V.A, Fiedler, T.J, Brew, K
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Base Sequence
Cattle
Circular Dichroism
Conserved Sequence
DNA Primers
Escherichia coli - enzymology
Escherichia coli - genetics
Galactosyltransferases - chemistry
Galactosyltransferases - metabolism
Humans
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Folding
Protein Structure, Secondary
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Alignment
Sequence Homology, Amino Acid
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Summary:The truncated catalytic domain of bovine beta1,4 galactosyltransferase-1 was expressed as inclusion bodies in E.coli and folded to generate 10-15 mg of active enzyme per liter of bacterial culture after extraction and purification under denaturing conditions. Mutations were introduced to investigate the roles of Trp312, Asp318, and Asp320, components of a highly conserved region of sequence in all known beta4GT-1 homologues that includes a cluster of acidic residues. Near and far UV CD spectra of the mutants indicate that the substitutions did not perturb the secondary and tertiary structure of beta4GT-1, and steady state kinetic studies indicate only minor effects on the response to an essential metal cofactor. However substitutions for the two aspartyl residues result in a reduction in catalytic efficiency of a magnitude that suggests they are important for catalysis. It seems possible that this anionic center may act in stabilizing a carbocation formed from the galactose component of the donor substrate in the transition state, reflecting a common reaction mechanism for beta-galactosyltransferase reactions.
ISSN:0959-6658
1460-2423