Pneumococcal beta-lactam resistance due to a conformational change in penicillin-binding protein 2x

Streptococcus pneumoniae is a life-threatening human pathogen that is increasingly resistant to a wide array of drugs. Resistance to beta-lactams, the most widely used antibiotics, is correlated with tens of amino acid substitutions in their targets; that is, the penicillin-binding proteins (PBPs), resulting from multiple events of recombination. To discriminate relevant substitutions from those that are incidental to the recombination process, we report the exhaustive characterization of all the mutations in the transpeptidase domain of PBP2x from the highly resistant strain 5204. A semi-automated method combining biochemical and microbiological approaches singled out 6 mutations of 41 (15%) that are essential for high level resistance. The hitherto uncharacterized I371T, R384G, M400T, and N605T together with the previously studied T338M and M339F account for nearly all the loss of affinity of PBP2x for beta-lactams. Most interestingly, I371T and R384G cause the conformational change of a loop that borders the entrance of the active site cavity, hampering antibiotic binding. For the first time all the mutations of a PBP relevant to beta-lactam resistance have been identified, providing new mechanistic insights. Most notable is the relationship between the decreased susceptibility to beta-lactams and the dynamic behavior of a loop.     

Dissection of a metal-ion-mediated conformational change in Tetrahymena ribozyme catalysis

Conformational changes are often required for the biological function of RNA molecules. In the Tetrahymena group I ribozyme reaction, a conformational change has been suggested to occur upon binding of the oligonucleotide substrate (S) or the guanosine nucleophile (G), leading to stronger binding of the second substrate. Recent work showed that the two substrates are bridged by a metal ion that coordinates both the nonbridging reactive phosphoryl oxygen of S and the 2'-OH of G. These results suggest that the energy from the metal ion substrate interactions is used to drive the proposed conformational change. In this work, we provide an experimental test for this model. The results provide strong support for the proposed conformational change and for a central role of the bridging metal ion in this change. The results from this work, combined with previous data, allow construction of a two-state model that quantitatively accounts for all of the observations in this and previous-work. This model provides a conceptual and quantitative framework that will facilitate understanding and further probing of the energetic and structural features of this conformational change and its role in catalysis.     

Kinetic mechanism of the beta-lactam synthetase of Streptomyces clavuligerus

Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.     

10-Formyltetrahydrofolate synthetase. Evidence for a conformational change in the enzyme upon binding of tetrahydropteroylpolyglutamates

The 10-formyltetrahydrofolate synthetase domain of the trifunctional enzyme C1-tetrahydrofolate synthase appears to undergo a conformational change in the presence of tetrahydropteroylpolyglutamates, MgATP, and ammonium ion. The binding of these ligands increases the denaturation temperature of the enzyme by 12 degrees C, abolishes the cold lability of the enzyme, and alters its susceptibility to digestion by chymotrypsin. The results suggest that a conformational change is dependent upon binding of the third glutamate residue of tetrahydropteroylpolyglutamates and the beta-phosphoryl group of MgATP. The Km values for MgATP and formate are lowered 3.6- and 520-fold, respectively, when tetrahydropteroyltriglutamate is used as the substrate in place of tetrahydropteroylmonoglutamate. A sensitive coupled assay involving C1-tetrahydrofolate synthase and serine hydroxymethyltransferase was developed to determine the activity of 10-formyltetrahydrofolate synthetase. The assay gives linear rates with the tetrahydropteroylpolyglutamates as substrates but not with the monoglutamate form.     

The mechanism of enhanced insulin amyloid fibril formation by NaCl is better explained by a conformational change model

The high propensity of insulin to fibrillate causes severe biomedical and biotechnological complications. Insulin fibrillation studies attain significant importance considering the prevalence of diabetes and the requirement of functional insulin in each dose. Although studied since the early years of the 20(th) century, elucidation of the mechanism of insulin fibrillation has not been understood completely. We have previously, through several studies, shown that insulin hexamer dissociates into monomer that undergoes partial unfolding before converting into mature fibrils. In this study we have established that NaCl enhances insulin fibrillation mainly due to subtle structural changes and is not a mere salt effect. We have carried out studies both in the presence and absence of urea and Gdn.HCl and compared the relationship between conformation of insulin induced by urea and Gdn.HCl with respect to NaCl at both pH 7.4 (hexamer) and pH 2 (monomer). Fibril formation was followed with a Thioflavin T assay and structural changes were monitored by circular dichroism and size-exclusion chromatography. The results show salt-insulin interactions are difficult to classify as commonly accepted Debye-Hückel or Hofmeister series interactions but instead a strong correlation between the association states and conformational states of insulin and their propensity to fibrillate is evident.     

Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the beta-lactam ring in Aspergillus nidulans

Genomic clones containing an Aspergillus nidulans isopenicillin N synthetase (IPNS) gene have been identified by heterologous hybridization with a Cephalosporium acremonium DNA probe. The open reading frame encodes a 331 amino acid polypeptide with extensive homology with the genes of other beta-lactam-producing fungi. The gene product has been overexpressed in Escherichia coli and shown to have activity of IPNS. This represents the first evidence at the molecular level that the biosynthesis of penicillins in A. nidulans occurs by the same pathway as in other beta-lactam-producing microorganisms. Comparison of available nucleotide sequences from IPNS genes suggests a horizontal transmission of the gene between the prokaryotic beta-lactam producers of the genus Streptomyces and the filamentous fungi.     

Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

Calcium is a universally employed cytosolic messenger in eukaryotic cells. Most of the proteins that bind signalling calcium are members of the calmodulin superfamily and share two or more helix-loop-helix motifs known as EF-hands. A model, based on structure comparison of different domains and supported by preliminary NMR data, has suggested that EF-hands involved in signal transduction undergo a major conformational change upon calcium binding from a 'closed' to an 'open' state allowing protein-protein interaction. We have determined the solution structures of the EF-hand pair from alpha-spectrin in the absence and in the presence of calcium. The structures are in the closed and open conformation respectively, providing a definite experimental proof for the closed-to-open model. Our results allow formulation of the rules which govern the movement induced by calcium. These rules may be generalized to other EF-hands since the key residues involved are conserved within the calmodulin family.     

A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity.

Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNA(Gln) binding to glnRS/ATP complex is absent in a noncognate tRNA tRNA(Glu)-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNA(Gln)-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.     

The effect of solvent viscosity on the rate-determining step of fatty acid synthetase

The overall activity of an animal fatty acid synthetase at the saturation level of substrate concentration decreased when the solvent viscosity, eta, of the reaction mixture was increased with viscogens such as glycerol, sucrose, and polyethylene glycol. The activity of the enzyme changed roughly proportional to eta-P, where p = 1.0 for glycerol, p = 0.66 for sucrose, and p less than 0.6 for polyethylene glycol with different molecular sizes. The thioesterase activity, which catalyzes the final partial reaction in the multifunctional enzyme, was not affected by 5-fold increase of solvent viscosity with sucrose. These results suggested that the rate-determining step of the enzyme other than the thioesterase reaction involves a microscopic transport step, the rate of which is influenced by the solvent viscosity. The microscopic transport step may be related to the transfer of the reaction intermediate from one active site to another or to the motion of a larger part of the enzyme requisite for the catalytic reaction. In the solution containing glycerol, the rate-determining motion was primarily diffusion limited since the inverse of the initial rate was proportional to eta, i.e., p = 1. Since the substrate concentration was at a saturation level in this experiment, the viscosity-dependent step cannot be the encounter between the enzyme and substrates, but must be intramolecular in origin, most probably the reaction catalyzed by beta-ketoacyl synthetase. In solutions containing other viscogens, however, p was less than 1.0, indicating a significant involvement of chemical steps in the rate-determining step as well. Bovine serum albumin, when used as a proteinic viscogen, also decreased the initial rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.     

Histidine modification and mutagenesis point to the involvement of a large conformational change in the mechanism of action of phage lambda lysozyme

Phage lambda lysozyme (lambdaL) is structurally related to other known lysozymes but its mechanism of action is different from the classical lysozyme mechanism, acting as a transglycosidase rather than a hydrolase. As two conformations have been revealed by the crystal structure, we investigated the effect of mutating and modifying a histidine located near to or far from the active site in the respective closed and open conformations. Whereas its asparagine mutation has little or no effect on activity, its N-carbethoxylation inactivates the enzyme. This provide further evidence for the involvement of the closed conformation and for the need of conformational mobility in lambdaL function.     

Structural and functional similarities in the ADP-forming amide bond ligase superfamily: implications for a substrate-induced conformational change in folylpolyglutamate synthetase

Comparison of the three-dimensional structures of folylpolyglutamate synthetase (FPGS) and the bacterial cell wall ligase UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD) reveals that these two enzymes have a remarkable structural similarity despite a low level of sequence identity. Both enzymes have a modular, multi-domain structure and catalyse a similar ATP-dependent reaction involving the addition of a glutamate residue to a carboxylate-containing substrate, tetrahydrofolate in the case of FPGS, and UDP-N-acetylmuramoyl-l-alanine in the case of MurD. Site-directed mutations of selected residues in the active site of Lactobacillus casei FPGS (P74A, E143A, E143D, E143Q, K185A, D313A, H316A, G411A and S412A) showed that most of these changes resulted in an almost complete loss of activity. Several of these amino acid residues in FPGS are found in structurally equivalent positions to active-site residues in MurD. Some insights into the function of these residues in FPGS activity are proposed, based on the roles surmised from the structures of two MurD. UDP-N-acetylmuramoyl-l-alanine.ADP complexes and a MurD. UDP-N-acetylmuramoyl-l-alanine-d-glutamate complex. Furthermore, the comparison has led us to propose that conformational changes induced by substrate binding in the reaction mechanism of FPGS result in a movement of the domains towards each other to more closely resemble the orientation of the corresponding domains in MurD. This relative domain movement may be a key feature of this new family of ADP-forming amide bond ligases.     

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.     

A diffusion Michaelis-Menten mechanism: continuous conformational change in enzymatic kinetics

We present a simple model which extends the Michaelis-Menten mechanism by incorporating a continuous protein conformational change in enzymatic catalysis. This model can represent a quantitative version for "rack" or "induced fit" mechanisms. In the steady-state it leads to an equation of the Michaelis-Menten form, but with the catalytic step at the active site showing strong dependence on solvent viscosity. We suggest that a careful examination of solvent viscosity effects on enzymatic activity may serve as a test for the conformational change hypothesis.