ABSTRACT
Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity.
Subject(s)
Bacterial Proteins , Carrier Proteins/genetics , Gene Expression , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/genetics , Peptidyl Transferases , Streptomyces/enzymology , Amino Acid Sequence , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cell Membrane/enzymology , Enzyme Stability , Hot Temperature , Molecular Sequence Data , Molecular Weight , Muramoylpentapeptide Carboxypeptidase/isolation & purification , Muramoylpentapeptide Carboxypeptidase/metabolism , Penicillin-Binding Proteins , Penicillins/metabolism , Plasmids , Sodium Chloride , Solubility , Streptomyces/genetics , Transformation, BacterialABSTRACT
The low-Mr penicillin-binding protein (PBP)/DD-transpeptidase of Streptomyces K15 is synthesized in the form of a 291-amino acid-residue precursor possessing a cleavable 29-amino acid-residue signal peptide. Sequence-similarity searches and hydrophobic-cluster analysis show that the Streptomyces K15 enzyme, the Escherichia coli PBPs/DD-carboxy-peptidases 5 and 6, the Bacillus subtilis PBP/DD-carboxypeptidase 5 and the spoIIA product (a putative PBP involved in the sporulation of B. subtilis) are structurally related and form a distinct class A of low-Mr PBPs/DD-peptidases. The distribution of the hydrophobic clusters along the amino acid sequences also shows that the Streptomyces K15 PBP, and by extension the other PBPs of class A, have similarity in the polypeptide folding, with the beta-lactamases of class A, with as reference the Streptomyces albus G and Staphylococcus aureus beta-lactamases of known three-dimensional structure. This comparison allows one to predict most of the secondary structures in the PBPs and the amino acid motifs that define the enzyme active sites.
