Dipeptide Binding to the Extended Active Site of the Streptomyces R61 d -Alanyl- d -alanine-peptidase: The Path to a Specific Substrate † (original) (raw)
Related papers
Biochemical Journal, 2003
The reactions between bacterial DD-peptidases and β-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific D-alanyl-D-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free Nterminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (D-γ Gln versus D-γ Glu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed.
Primary structure of the Streptomyces R61 extracellular DD‐peptidase
European Journal of Biochemistry, 1987
In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD‐peptidase was fragmented by (a) cyanogen bromide cleavage of the carboxymethylated protein, (b) trypsin digestion of the carboxymethylated protein and (c) trypsin digestion of the protein treated with β‐iodopenicillinate and endoxo‐δ4‐tetrahydrophthalic acid. The isolated peptides, which altogether represented more than 50% of the polypeptide chain, were sequenced. The data thus obtained were in excellent agreement with the primary structure of the protein as deduced from the nucleotide sequence of the cloned gene. Though a weak acylating agent, β‐iodopenicillanate reacted selectively with the active site of the DD‐peptidase and formed an adduct which mas much more stable than that formed with benzylpenicillin, thus facilitating the isolation and characterization of the active‐site peptide.
Protein Science, 2005
The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COOgroup has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibilty of the transpeptidation reaction. .
Biochemical Journal, 1993
Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding was also drastically decreased, except for the reactions with nitrocefin and methicillin. The consequences of the first mutation were more surprising, since a large proportion of the thiolesterase activity was retained, together with the penicillin-binding capacity. Conversely, the peptidase properties was severely affected. In both cases, a drastic decrease in the transferase activity was observed. The results are compared with those obtained by mutation of the corresponding residue in the class A beta-lactamase of Streptomyces albus G.
Inactivation of Bacterial dd -Peptidase by β-Sultams †
Biochemistry, 2005
N-Acyl--sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to -sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the -sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located. D-Alanyl-D-alanine carboxypeptidase/transpeptidases [DDpeptidases and also known as penicillin-binding proteins, or PBPs (1)] are enzymes produced by bacteria that catalyze the final steps of their cell wall biosynthesis (1, 2). Bacteria, with a very few exceptions, have a cross-linked cell wall, the formation of which involves a transpeptidation reaction that is catalyzed by bacterial transpeptidases/PBPs. The PBPs have been classified into two groups: the low molecular mass (LMM) PBPs and the high molecular mass (HMM) PBPs (3). The low molecular mass group includes -lactamases that catalyze the hydrolysis of -lactam antibiotics, the D-Ala-D-Ala carboxypeptidases, and endopeptidases. The HMM PBPs catalyze the transpeptidation reaction that cross-links the glycan strands of the cell wall (3, 4).
The precursor of the Streptomyces R61 DD-peptidase containing a C-terminal extension is inactive
FEBS Letters, 1994
The Streptomyces R6l DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein.The introduction of a 'stop' codon after that corresponding to the C-terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli.