RESUMEN
The resistance of bacteria to ß-lactam antibiotics is primarily caused by the production of ß-lactamases. Here, novel crystal structures of the native ß-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical ß-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published ß-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new ß-lactamase inhibitors.
Asunto(s)
Ácido Penicilánico , beta-Lactamasas , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ácido Penicilánico/química , Ácido Penicilánico/metabolismo , Ácido Penicilánico/farmacología , Tazobactam , beta-Lactamasas/químicaRESUMEN
Enzymes conjugated to magnetic nanoparticles (MNPs) undergo changes in the catalytic activity of the non-heating low-frequency magnetic field (LFMF). We apply in silico simulations by molecular dynamics (MD) and in vitro spectroscopic analysis of the enzyme kinetics and secondary structure to study α-chymotrypsin (CT) conjugated to gold-coated iron oxide MNPs. The latter are functionalized by either carboxylic or amino group moieties to vary the points of enzyme attachment. The MD simulation suggests that application of the stretching force to the CT globule by its amino or carboxylic groups causes shrinkage of the substrate-binding site but little if any changes in the catalytic triad. Consistent with this, in CT conjugated to MNPs by either amino or carboxylic groups, LFMF alters the Michaelis-Menten constant but not the apparent catalytic constant k cat (= V max/[E]o). Irrespective of the point of conjugation to MNPs, the CT secondary structure was affected with nearly complete loss of α-helices and increase in the random structures in LFMF, as shown by attenuated total reflection Fourier transformed infrared spectroscopy. Both the catalytic activity and the protein structure of MNP-CT conjugates restored 3 h after the field exposure. We believe that such remotely actuated systems can find applications in advanced manufacturing, nanomedicine, and other areas.
RESUMEN
BACKGROUND: GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis. METHODS: The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74cat) and Myceliophthora thermophila (MtXeg74A) were carried out. RESULTS: MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between Cα atoms of aromatic residues involved in xyloglucan binding in -3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10-12 Å. For PoGH74cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft. CONCLUSIONS: These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity. GENERAL SIGNIFICANCE: Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Paenibacillus/enzimología , Sordariales/enzimología , Dominio Catalítico , Glucanos/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Simulación de Dinámica Molecular , Mutación , Paenibacillus/genética , Paenibacillus/metabolismo , Conformación Proteica , Dominios Proteicos , Sordariales/genética , Sordariales/metabolismo , Xilanos/metabolismoRESUMEN
Bacterial resistance to ß-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed ß-lactamases (ßLs). The evolution of serine-class A ß-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme's active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM-type ß-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl-enzyme complex and the hydrolysis of the ß-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type ß-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of ß-lactamases.
Asunto(s)
Bacterias/enzimología , Biocatálisis , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Farmacorresistencia Bacteriana , Conformación ProteicaRESUMEN
Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.
Asunto(s)
Quimotripsina/metabolismo , Biocatálisis , Quimotripsina/química , Oro/química , Campos Magnéticos , Nanopartículas de Magnetita/química , Modelos MolecularesRESUMEN
Highly mutable ß-lactamases are responsible for the ability of Gram-negative bacteria to resist ß-lactam antibiotics. Using site-directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM-type ß-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) ß-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of ß-lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of ß-lactamase TEM-1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of ß-lactamase TEM-1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N-terminal α-helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of ß-lactamases can be used as a new potential target for inhibiting the enzyme.
RESUMEN
ß-Сyclodextrin (CD) is a perspective class of excipients used in pharmaceutical formulations to enhance solubility, bioavailability, and pharmacokinetics of various poorly soluble drugs, forming a non-covalent guest-host complex. However, the development of such formulations is usually a very laborious and time-consuming process due to lack of appropriate analytical tools to directly track and study the detailed molecular mechanism of such complex formation. Here, using guest-host complexes of fluoroquinolones (FQ) with CDs, as an example, we demonstrate the utility of ATR-FTIR to determine the thermodynamic stability, as well as structural features associated with complex formation, including involvement of certain functional groups. Furthermore, varying the CD's side groups, we were able to tailor the CD's geometry and binding surface to make FQ-CD interactions strong enough to potentially affect its pharmacokinetics and justify development of a new sustained-release drug formulation (dissociation constant decreased from 5 * 10-3 M to 10-5 M). 3D molecular modeling with energy optimization supports the findings and conclusions made on the basis of ATR-FTIR data analysis and explains the observed difference in dissociation constants.
Asunto(s)
Antibacterianos/química , Excipientes/química , Fluoroquinolonas/química , beta-Ciclodextrinas/química , Cristalización , Concentración de Iones de Hidrógeno , Levofloxacino/química , Modelos Moleculares , Espectroscopía Infrarroja por Transformada de Fourier , Termodinámica , Difracción de Rayos XRESUMEN
In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability.
Asunto(s)
Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Mutagénesis Sitio-Dirigida , Penicillium/enzimología , Temperatura , Secuencia de Aminoácidos , Endo-1,4-beta Xilanasas/química , Estabilidad de Enzimas , Modelos Moleculares , Mutación , Penicillium/genética , Conformación ProteicaRESUMEN
BACKGROUND: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. METHODS/PRINCIPAL FINDINGS: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. CONCLUSIONS: Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.
Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Anticuerpos Monoclonales , Células Endoteliales/metabolismo , Epidídimo/metabolismo , Mapeo Epitopo , Humanos , Pulmón/metabolismo , Masculino , Próstata/metabolismo , Semen/metabolismoRESUMEN
The dipole interaction model is a classical electromagnetic theory for calculating circular dichroism (CD) resulting from the π-π* transitions of amides. The theoretical model, pioneered by J. Applequist, is assembled into a package, DInaMo, written in Fortran allowing for treatment of proteins. DInaMo reads Protein Data Bank formatted files of structures generated by molecular mechanics or reconstructed secondary structures. Crystal structures cannot be used directly with DInaMo; they either need to be rebuilt with idealized bond angles and lengths, or they need to be energy minimized to adjust bond lengths and bond angles because it is common for crystal structure geometries to have slightly short bond lengths, and DInaMo is sensitive to this. DInaMo reduces all the amide chromophores to points with anisotropic polarizability and all nonchromophoric aliphatic atoms including hydrogens to points with isotropic polarizability; all other atoms are ignored. By determining the interactions among the chromophoric and nonchromophoric parts of the molecule using empirically derived polarizabilities, the rotational and dipole strengths are determined leading to the calculation of CD. Furthermore, ignoring hydrogens bound to methyl groups is initially explored and proves to be a good approximation. Theoretical calculations on 24 proteins agree with experiment showing bands with similar morphology and maxima.