title: Domain organization of penicillin-binding protein 5 from Escherichia coli analysed by C-terminal truncation.
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.
authors.affiliation: Department of Biochemistry, University of Groningen, The Netherlands.
authors.forename: M P
authors.initials: MP
authors.lastname: van der Linden
authors.forename: L
authors.initials: L
authors.lastname: de Haan
authors.forename: W
authors.initials: W
authors.lastname: Keck
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000235:genetics; D006602:Hexosyltransferases; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003062:Codon; D009838:Oligodeoxyribonucleotides; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase