ABSTRACT
Five class A beta-lactamases produced by various mesophilic bacterial species have been compared. Although closely related in primary and overall structures, these enzymes exhibit very different stabilities. In order to investigate the factors responsible for these differences, several features deduced from the amino acid composition and three-dimensional structures were studied for the five proteins. This analysis revealed that higher stability appeared to correlate with increased numbers of intramolecular hydrogen bonds and of salt bridges. By contrast, the global hydrophobicity of the protein seemed to play a relatively minor role. A strongly unfavourable balance between charged residues and the presence of a cis-peptide bond preceding a non-proline residue might also contribute to the particularly low stability of two of the enzymes.
Subject(s)
Enzyme Stability/genetics , beta-Lactamases/chemistry , Amino Acid Sequence , Amino Acids/analysis , Bacteria/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Crystallography , Enzyme Stability/physiology , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Fungi/enzymology , Hydrogen Bonding , Molecular Sequence Data , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Temperature , Thermodynamics , beta-Lactamases/classification , beta-Lactamases/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins , Carrier Proteins/genetics , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/genetics , Penicillins/metabolism , Peptidyl Transferases/genetics , Streptomyces/enzymology , Amino Acid Sequence , Amino Acids/analysis , Base Sequence , DNA Probes , Molecular Sequence Data , Oligonucleotide Probes , Open Reading Frames , Penicillin-Binding Proteins , Plasmids , Restriction Mapping , Sequence Homology, Nucleic Acid , Staphylococcus aureus/enzymology , beta-Lactamases/geneticsABSTRACT
The gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G has been cloned in Escherichia coli DH5 alpha MCR via pBR322 or 325, and then transferred into Streptomyces lividans TK24 via pIJ486, with substantial amplification of the expressed DD-peptidase. The gene has the information for the synthesis of a 255 amino acid precursor, the amino terminal region of which has the characteristic features of a signal peptide. The primary structure as deduced from nucleotide sequencing confirms that previously determined by chemical methods except for the occurrence of an Asp instead of Asn at position 1 and an additional Ala immediately downstream of Pro67.
Subject(s)
Muramoylpentapeptide Carboxypeptidase/genetics , Streptomyces/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Enzyme Precursors/biosynthesis , Enzyme Precursors/genetics , Escherichia coli/genetics , Molecular Sequence Data , Muramoylpentapeptide Carboxypeptidase/biosynthesis , Muramoylpentapeptide Carboxypeptidase/metabolism , Protein Sorting Signals/genetics , Streptomyces/enzymologyABSTRACT
The gene encoding the extracellular, active-site serine beta-lactamase of Actinomadura R39, previously cloned into Streptomyces lividans, has the information for the synthesis of a 304 amino acid protein, the amino terminal region of which has the characteristic features of a signal peptide. The Actinomadura R39 beta-lactamase is another member of the class A beta-lactamases. In particular, it shows high homology with the beta-lactamase of Bacillus licheniformis.
