Cloning and expression of the beta-N-acetylglucosaminidase gene from Streptococcus pneumoniae. Generation of truncated enzymes with modified aglycon specificity.
The gene encoding a beta-N-acetylglucosaminidase from Streptococcus pneumoniae has been obtained by screening an expression library for beta-N-acetylglucosaminidase activity. Clones of different nucleotide sizes each having arylglycoside activity were obtained, and DNA sequencing revealed a gene of 3933 base pairs possessing typical bacterial transcription initiation and termination sequences and terminating in an ochre stop codon. Computer analysis of the translated protein of 1311 amino acids (144,210 Da) identified a tandem repeat within which lies a sequence homologous with six other hexosaminidase gene products from a wide variety of species ranging from bacteria to humans. Also found were an amino-terminal putative secretion signal peptide and a carboxyl-terminal cell sorting/anchorage motif typically found in over 20 other Gram-positive surface proteins. The expression of an almost complete DNA clone in Escherichia coli produced a functional and authentic beta-N-acetylglucosaminidase with aglycon specificity identical to the wild-type enzyme. However, enzymes produced from truncated DNA clones show more restricted aglycon specificity and are unable to hydrolyze terminal beta 1-2GlcNAc residues from N-glycans containing a bisecting N-acetylglucosamine. The availability of these clones allows structural analyses to be made of catalytic and oligosaccharide recognition protein domains that enhance functional activity.
Clarke
V A
VA
Department of Biochemistry, University of Oxford, United Kingdom.
Platt
N
N
Butters
T D
TD
D000118:Acetylglucosaminidase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013402:Sugar Alcohols; D000118:Acetylglucosaminidase