7 research outputs found
Crystal structure of a complex between the Actinomadura R39 DD-peptidase and a peptidoglycan-mimetic boronate inhibitor: interpretation of a transition state analogue in terms of catalytic mechanism.
peer reviewedThe Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small D-alanyl-D-alanine terminating peptides, especially those with a side chain that mimics the amino terminus of the stem peptide precursor to the bacterial cell wall. This paper describes the synthesis of (D-alpha-aminopimelylamino)-D-1-ethylboronic acid, designed to be a peptidoglycan-mimetic transition state analogue inhibitor of the R39 DD-peptidase. The boronate was found to be a potent inhibitor of the peptidase with a K(i) value of 32 +/- 6 nM. Since it binds some 30 times more strongly than the analogous peptide substrate, the boronate may well be a transition state analogue. A crystal structure of the inhibitory complex shows the boronate covalently bound to the nucleophilic active site Ser 49. The aminopimelyl side chain is bound into the site previously identified as specific for this moiety. One boronate oxygen is held in the oxyanion hole; the other, occupying the leaving group site of acylation or the nucleophile site of deacylation, appears to be hydrogen-bonded to the hydroxyl group of Ser 298. The Ser 49 oxygen appears to be hydrogen bonded to Lys 52. If it is assumed that this structure does resemble a high-energy tetrahedral intermediate in catalysis, it seems likely that Ser 298 participates as part of a proton transfer chain initiated by Lys 52 or Lys 410 as the primary proton donor/acceptor. The structure, therefore, supports a particular class of mechanism that employs this proton transfer device
Interactions of “Bora-Penicilloates” with Serine β‑Lactamases and DD-Peptidases
Specific
boronic acids are generally powerful tetrahedral intermediate/transition
state analogue inhibitors of serine amidohydrolases. This group of
enzymes includes bacterial β-lactamases and DD-peptidases where
there has been considerable development of boronic acid inhibitors.
This paper describes the synthesis, determination of the inhibitory
activity, and analysis of the results from two α-(2-thiazolidinyl)
boronic acids that are closer analogues of particular tetrahedral
intermediates involved in β-lactamase and DD-peptidase catalysis
than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic
acid, is a direct analogue of the deacylation tetrahedral intermediates
of these enzymes. These compounds are micromolar inhibitors of class
C β-lactamases but, very unexpectedly, not inhibitors of class
A β-lactamases. We rationalize the latter result on the basis
of a new mechanism of boronic acid inhibition of the class A enzymes.
A stable inhibitory complex is not accessible because of the instability
of an intermediate on its pathway of formation. The new boronic acids
also do not inhibit bacterial DD-peptidases (penicillin-binding proteins).
This result strongly supports a central feature of a previously proposed
mechanism of action of β-lactam antibiotics, where deacylation
of β-lactam-derived acyl-enzymes is not possible because of
unfavorable steric interactions
4-Quinolones as Noncovalent Inhibitors of High Molecular Mass Penicillin-Binding Proteins
Penicillin-binding proteins (PBPs) are important bacterial
enzymes
that carry out the final steps of bacterial cell wall assembly. Their
DD-transpeptidase activity accomplishes the essential peptide cross-linking
step of the cell wall. To date, all attempts to discover effective
inhibitors of PBPs, apart from β-lactams, have not led to new
antibiotics. Therefore, the need for new classes of efficient inhibitors
of these enzymes remains. Guided by a computational fragment-based
docking procedure, carried out on <i>Escherichia coli</i> PBP5, we have designed and synthesized a series of 4-quinolones
as potential inhibitors of PBPs. We describe their binding to the
PBPs of <i>E. coli</i> and <i>Bacillus subtilis</i>. Notably, these compounds bind quite tightly to the essential high
molecular mass PBPs
Inhibition of dd-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the <i>Actinomadura</i> R39 dd-Peptidase
Inhibitors
of bacterial dd-peptidases represent potential antibiotics.
In the search for alternatives to β-lactams, we have investigated
a series of compounds designed to generate transition state analogue
structures upon reaction with dd-peptidases. The compounds
contain a combination of a peptidoglycan-mimetic specificity handle
and a warhead capable of delivering a tetrahedral anion to the enzyme
active site. The latter includes a boronic acid, two alcohols, an
aldehyde, and a trifluoroketone. The compounds were tested against
two low-molecular mass class C dd-peptidases. As expected
from previous observations, the boronic acid was a potent inhibitor,
but rather unexpectedly from precedent, the trifluoroketone [d-α-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also
very effective. Taking into account competing hydration, we found
the trifluoroketone was the strongest inhibitor of the <i>Actinomadura</i> R39 dd-peptidase, with a subnanomolar (free ketone) inhibition
constant. A crystal structure of the complex between the trifluoroketone
and the R39 enzyme showed that a tetrahedral adduct had indeed formed
with the active site serine nucleophile. The trifluoroketone moiety,
therefore, should be considered along with boronic acids and phosphonates
as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics
Inhibition of dd-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 dd-Peptidase.
Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-alpha-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics
Spécificité de substrat des DD-peptidases bactériennes de faible poids moléculaire
The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and lowmolecular mass (LMM) enzymes. The latter group, which is subdivided into classes A−C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined.
This paper describes the steady-state kinetics of hydrolysis of a series of specific
peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 DD-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be
able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown