PMID:8424800
| Citation |
van der Linden, MP, de Haan, L and Keck, W (1993) Domain organization of penicillin-binding protein 5 from Escherichia coli analysed by C-terminal truncation. Biochem. J. 289 ( Pt 2):593-8 |
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| Abstract |
The structural organization of penicillin-binding protein (PBP) 5 was investigated by C-terminal truncation. Compared with other low-M(r) penicillin-interacting proteins, PBP5 carries a C-terminal extension of about 100 amino acids. The sites for introduction of stop codons were chosen on the basis of the established three-dimensional structure of the Streptomyces albus G beta-lactamase [Dideberg, Charlier, Wéry, Dehottay, Dusart, Erpicum, Frère and Ghuysen (1987) Biochem. J. 245, 911-913] and comparative hydrophobic cluster analysis [Gaboriaud, Bissery, Bencheritt and Mornon (1987) FEBS Lett. 224, 149-155]. Two stop codons were introduced at positions Ile-354 or Val-348 to construct an optimized soluble form of PBP5 for crystallization purposes. The newly constructed soluble and enzymically active form (PBP5s353) was isolated by dye-affinity chromatography and gave rise to small crystals. Another two stop codons were introduced at positions Arg-261 or Ala-276 to determine the minimal enzymically active 'core protein'. The truncated form (PBP5s275), missing the entire C-terminal extension, showed unaltered penicillin-binding characteristics and a catalytic-centre activity 40% that of PBP5s353 + 9 using bisacetyl-L-Lys-D-Ala-D-Ala as substrate. This protein, however was more susceptible to proteolytic degradation, which might indicate a role of the C-terminal portion in stabilizing the protein. |
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| Keywords |
Amino Acid Sequence; Antibodies; Bacillus subtilis/genetics; Bacterial Proteins; Base Sequence; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cloning, Molecular/methods; Codon; Escherichia coli/genetics; Geobacillus stearothermophilus/genetics; Hexosyltransferases; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Weight; Muramoylpentapeptide Carboxypeptidase/chemistry; Muramoylpentapeptide Carboxypeptidase/genetics; Muramoylpentapeptide Carboxypeptidase/metabolism; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Penicillin-Binding Proteins; Penicillins/metabolism; Peptidyl Transferases; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid |
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