ABSTRACT
Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.
Subject(s)
Pyruvate Kinase , Zymomonas , Humans , Zymomonas/enzymology , Zymomonas/genetics , Zymomonas/chemistry , Zymomonas/metabolism , Pyruvate Kinase/chemistry , Pyruvate Kinase/metabolism , Pyruvate Kinase/genetics , Amino Acid Substitution , Protein Stability , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Enzyme Stability , Liver/enzymology , Liver/metabolism , Liver/chemistry , Phosphoenolpyruvate/metabolism , Phosphoenolpyruvate/chemistryABSTRACT
Understanding the structural arrangements of protein oligomers can support the design of ligands that interfere with their function in order to develop new therapeutic concepts for disease treatment. Recent crystallographic studies have elucidated a novel twisted and functionally inactive form of the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative target in the fight against shigellosis. Active-site ligands have been identified that stimulate the rearrangement of one monomeric subunit by 130° against the other one to form an inactive twisted homodimer state. To assess whether the crystallographic observations also reflect the conformation in solution and rule out effects from crystal packing, we performed 19F-NMR spectroscopy with the introduction of 5-fluorotryptophans at four sites in TGT. The inhibitor-induced conformation of TGT in solution was assessed based on 19F-NMR chemical shift perturbations. We investigated the effect of C(4) substituted lin-benzoguanine ligands and identified a correlation between dynamic protein rearrangements and ligand-binding features in the corresponding crystal structures. These involve the destabilization of a helix next to the active site and the integrity of a flexible loop-helix motif. Ligands that either completely lack an attached C(4) substituent or use it to stabilize the geometry of the functionally competent dimer state do not indicate the presence of the twisted dimer form in the NMR spectra. The perturbation of crucial structural motifs in the inhibitors correlates with an increasing formation of the inactive twisted dimer state, suggesting these ligands are able to shift a conformational equilibrium from active C2-symmetric to inactive twisted dimer conformations. These findings suggest a novel concept for the design of drug candidates for further development.
Subject(s)
Zymomonas , Catalytic Domain , Crystallography, X-Ray , Guanine/metabolism , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Pentosyltransferases/chemistry , Protein Conformation , RNA, Transfer/chemistry , Zymomonas/chemistryABSTRACT
BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20â keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659â keV, the monochromated beam is focused to 4.1â µm (V) × 8.5â µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012â photonsâ s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.
Subject(s)
Crystallography, X-Ray/instrumentation , Macromolecular Substances/chemistry , Proteins/chemistry , Carbon Radioisotopes , Equipment Design , Legionella/chemistry , Muramidase/chemistry , Neisseria meningitidis/chemistry , Protein Structural Elements , Synchrotrons , Zymomonas/chemistryABSTRACT
Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.
Subject(s)
Disulfides/chemistry , Pentosyltransferases/chemistry , Small Molecule Libraries/pharmacology , Zymomonas/enzymology , Binding Sites/drug effects , Ligands , Models, Molecular , Protein Multimerization/drug effects , Small Molecule Libraries/chemistry , Zymomonas/chemistryABSTRACT
Fructan based biopolymers have been extensively characterized and explored for their potential applications. Linear chained biopolymers, like levan-type fructan, have gained attention because they have exhibited unconventional stretchable and unbendable properties along with biodegradable and biocompatible nature. Current study deals with the chemical characterization and cytotoxic analysis of fructose based exopolysaccharide that was extracellularly produced by an indigenously isolated bacterial species (Zymomonas mobilis KIBGE-IB14). Maximum yield of exopolysaccharide (44.7 gL-1) was attained after 72 h of incubation at 30 °C under shaking conditions (180 rpm) when the culture medium was supplemented with 150.0 gL-1 of sucrose as a sole carbon source. This exopolysaccharide displayed high water solubility index (96.0%) with low water holding capacity (17.0%) and an intrinsic viscosity of about 0.447 dL g-1. This biopolymer exhibited a characteristic linear homopolysaccharide structure of levan when characterized using Fourier Transform Infrared (FTIR), Nuclear Magnetic Resonance (NMR) spectroscopy (1H, 13C, TOCSY and NOESY) while, Atomic Force Microscopy (AFM) revealed its pointed and thorny structure. The decomposition temperature of levan was approximately 245 °C as revealed by Thermal Gravimetric Analysis (TGA). X-Ray Diffraction (XRD) results revealed its amorphous nature with crystalline phase. Cytotoxicity of different concentrations of levan was investigated against mouse fibroblast cell lines by measuring their cellular metabolic activity and it was noticed that a higher concentration of levan (2.0 mg ml-1) permitted the normal cell growth of NIH/3T3 cell lines. This non-cytotoxic and biocompatible nature suggests that this levan has the capability to be utilized in food and drug-based formulations as it exhibited biomedical potential.
Subject(s)
Biopolymers/pharmacology , Fructans/pharmacology , Zymomonas/chemistry , Animals , Biopolymers/biosynthesis , Biopolymers/chemistry , Carbohydrate Conformation , Cell Survival/drug effects , Dose-Response Relationship, Drug , Fructans/biosynthesis , Fructans/chemistry , Mice , NIH 3T3 Cells , Zymomonas/metabolismABSTRACT
Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.
Subject(s)
Pentosyltransferases/chemistry , Zymomonas/enzymology , Catalytic Domain , Crystallography, X-Ray , Enzyme Stability , Models, Molecular , Mutation , Pentosyltransferases/genetics , Protein Conformation , Protein Multimerization , Zymomonas/chemistry , Zymomonas/geneticsABSTRACT
The bioproduction of lactobionic acid and its salts can be performed by enzymatic complex glucose-fructose oxidoreductase (GFOR) and glucono-δ-lactonase (GL) of Zymomonas mobilis. Considering the applicability of these compounds in pharmaceutical area, the aim of this study was to assess the accelerated and long-term stability studies of sodium, potassium, calcium lactobionate, and lactobionic acid. Furthermore, stress tests were performed to evaluate the stability against pH, temperature and oxidation. The samples submitted to degradation tests were analyzed by high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry analysis (HRMS-ESI-QTOF). Sodium, potassium, and calcium lactobionate were stable for six months of analyses considering the accelerated (40 °C and 75% RH) and long-term (30 °C and 75% RH) stability studies. The presence of lactobiono-δ-lactone and a significant increase in moisture were observed for both biosynthesized and commercially available lactobionic acid samples. Against the forced degradation tests, all the lactobionate salts and lactobionic acid showed to be stable upon alkaline and acid pH conditions, at 60 and 80 °C, and also against UV light exposition. Furthermore, the presence of lactobiono-δ-lactone form was observed in lactobionic acid samples. However, the degradation of both lactobionic acid and lactobionate salts was evident in the presence of hydrogen peroxide. This degradation kinetic profile suggests, that lactobionate salts follows a zero-order reaction model and lactobionic acid follows a second-order kinetic. The MS analysis of the main degradation product suggests a molecular formula C11H20O10 resulting from the oxidative decarboxylation. This report brings an amount of results as contribution to the scarce information regarding the chemical and physical-chemical stability of sodium, potassium, calcium lactobionate, and lactobionic acid. These data may be useful and serve as reference, in view of the multipurpose applications of the cited compounds.
Subject(s)
Calcium/chemistry , Disaccharides/chemistry , Potassium/chemistry , Sodium/chemistry , Zymomonas/chemistry , Bioreactors , Decarboxylation , Drug Stability , Hydrogen Peroxide/chemistry , Hydrogen-Ion Concentration , Kinetics , Lactones , Mass Spectrometry , Oxidative Stress , Oxidoreductases/chemistry , Oxygen/chemistry , Temperature , Ultraviolet RaysABSTRACT
Lignocellulosic biomass is a sustainable feedstock for fuel ethanol production, but it is characterized by low mass and energy densities, and distributed production with relatively small scales is more suitable for cellulosic ethanol, which can better balance cost for the feedstock logistics. Lignocellulosic biomass is recalcitrant to degradation, and pretreatment is needed, but more efficient pretreatment technologies should be developed based on an in-depth understanding of its biosynthesis and regulation for engineering plant cell walls with less recalcitrance. Simultaneous saccharification and co-fermentation has been developed for cellulosic ethanol production, but the concept has been mistakenly defined, since the saccharification and co-fermentation are by no means simultaneous. Lignin is unreactive, which not only occupies reactor spaces during the enzymatic hydrolysis of the cellulose component and ethanol fermentation thereafter, but also requires extra mixing, making high solid loading difficult for lignocellulosic biomass and ethanol titers substantially compromised, which consequently increases energy consumption for ethanol distillation and stillage discharge, presenting another challenge for cellulosic ethanol production. Pentose sugars released from the hydrolysis of hemicelluloses are not fermentable with Saccharomyces cerevisiae used for ethanol production from sugar- and starch-based feedstocks, and engineering the brewing yeast and other ethanologenic species such as Zymomonas mobilis with pentose metabolism has been performed within the past decades. However strategies for the simultaneous co-fermentation of pentose and hexose sugars that have been pursued overwhelmingly for strain development might be modified for robust ethanol production. Finally, unit integration and system optimization are needed to maximize economic and environmental benefits for cellulosic ethanol production. In this article, we critically reviewed updated progress, and highlighted challenges and strategies for solutions.
Subject(s)
Biotechnology/methods , Ethanol/chemistry , Lignin/chemistry , Biomass , Bioreactors , Carbohydrate Metabolism , Cellulose/chemistry , Ethanol/metabolism , Hydrolysis , Lignin/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Zymomonas/chemistry , Zymomonas/metabolismABSTRACT
The intestinal disease shigellosis caused by Shigella bacteria affects over 120â million people annually. There is an urgent demand for new drugs as resistance against common antibiotics emerges. Bacterial tRNA-guanine transglycosylase (TGT) is a druggable target and controls the pathogenicity of Shigella flexneri. We report the synthesis of sugar-functionalized lin-benzoguanines addressing the ribose-33 pocket of TGT from Zymomonas mobilis. Ligand binding was analyzed by isothermal titration calorimetry and X-ray crystallography. Pocket occupancy was optimized by variation of size and protective groups of the sugars. The participation of a polycyclic water-cluster in the recognition of the sugar moiety was revealed. Acetonide-protected ribo- and psicofuranosyl derivatives are highly potent, benefiting from structural rigidity, good solubility, and metabolic stability. We conclude that sugar acetonides have a significant but not yet broadly recognized value in drug development.
Subject(s)
Guanine/chemistry , Pentosyltransferases/chemistry , RNA, Transfer/chemistry , Ribose/chemistry , Sugars/chemistry , Zymomonas/chemistry , Crystallography, X-Ray , Molecular Structure , Pentosyltransferases/metabolism , Protein Binding , SolventsABSTRACT
Currently, the power and usefulness of biocatalysis in organic synthesis is undeniable, mainly due to the very high enantiomeric excess reached using enzymes, in an attempt to emulate natural processes. However, the use of isolated enzymes has some significant drawbacks, the most important of which is cost. The use of whole cells has emerged as a useful strategy with several advantages over isolated enzymes; for this reason, modern research in this field is increasing, and various reports have been published recently. This review surveys the most recent developments in the enantioselective reduction of carbon-carbon double bonds and prochiral ketones and the oxidation of prochiral sulfides using whole cells as biocatalytic systems.
Subject(s)
Alcohols/chemistry , Ketones/chemistry , Penicillium/chemistry , Saccharomyces cerevisiae/chemistry , Sulfides/chemistry , Zymomonas/chemistry , Alcohols/metabolism , Biocatalysis , Biotransformation , Ketones/metabolism , Oxidation-Reduction , Penicillium/enzymology , Saccharomyces cerevisiae/enzymology , Stereoisomerism , Sulfides/metabolism , Zymomonas/enzymologyABSTRACT
Electronic absorption spectra are oftentimes used to identify reaction intermediates or substrates/products in enzymatic systems, as long as absorption bands can be unequivocally assigned to the species being studied. The latter task is far from trivial given the transient nature of some states and the complexity of the surrounding environment around the active site. To identify unique spectral fingerprints, controlled experiments with model compounds have been used in the past, but even these can sometimes be unreliable. Circular dichroism (CD) and ultraviolet-visible spectra have been tools of choice in the study of the rich chemistry of thiamin diphosphate-dependent enzymes. In this study, we focus on the Zymomonas mobilis pyruvate decarboxylase, and mutant analogues thereof, as a prototypical representative of the thiamin diphosphate (ThDP) enzyme superfamily. Through the use of electronic structure methods, we analyze the nature of electronic excitations in the cofactor. We find that all the determining CD bands around the 280-340 nm spectral range correspond to charge-transfer excitations between the pyrimidine and thiazolium rings of ThDP, which, most likely, is a general property of related ThDP-dependent enzymes. While we can confirm the assignments of previously proposed bands to chemical states, our calculations further suggest that a hitherto unassigned band of enzyme-bound ThDP reports on the ionization state of the canonical glutamate that is required for cofactor activation. This finding expands the spectroscopic "library" of chemical states of ThDP enzymes, permitting a simultaneous assignment of both the cofactor ThDP and the activating glutamate. We anticipate this finding to be helpful for mechanistic analyses of related ThDP enzymes.
Subject(s)
Bacterial Proteins/chemistry , Coenzymes/chemistry , Glutamic Acid/chemistry , Pyruvate Decarboxylase/chemistry , Thiamine Pyrophosphate/chemistry , Zymomonas/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Coenzymes/metabolism , Electron Transport , Gene Expression , Glutamic Acid/metabolism , Kinetics , Molecular Dynamics Simulation , Mutation , Protein Structure, Secondary , Pyrimidines/chemistry , Pyruvate Decarboxylase/genetics , Pyruvate Decarboxylase/metabolism , Static Electricity , Thermodynamics , Thiamine Pyrophosphate/metabolism , Zymomonas/enzymologyABSTRACT
This work reports a novel method of recovering anthocyanin compounds from highly-pigmented grapes via a fermentation based approach. It was hypothesized that batch growth of Zymomonas mobilis on simple medium would produce both ethanol and enzymes/biomass-acting materials, the combination of which will provide a superior extraction when compared to simple alcohol extraction. To examine this hypothesis, Z. mobilis was fermented in a batch consisting of mashed Vitis vinifera and glucose, and the recovered anthocyanin pool was compared to that recovered via extraction with ethanol. Data indicated higher amounts of anthocyanins were recovered when compared to simple solvent addition. Additionally, the percent polymeric form of the anthocyanins could be manipulated by the level of aeration maintained in the fermentation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:601-605, 2016.
Subject(s)
Anthocyanins/isolation & purification , Anthocyanins/metabolism , Ethanol/metabolism , Vitis/metabolism , Zymomonas/metabolism , Anthocyanins/chemistry , Ethanol/chemistry , Fermentation , Vitis/chemistry , Zymomonas/chemistry , Zymomonas/growth & developmentABSTRACT
Zymomonas mobilis immobilized in doped calcium alginate (Ca-alginate) was successfully employed for the production of ethanol in an immobilized cell reactor. Polyethylene oxide and F127 dimethacrylate were evaluated as potential dopants for Ca-alginate beads to decrease lag time and increase initial ethanol yield. The influence of the type and concentration of the dopant on the effectiveness of the microbe immobilized in Ca-alginate beads to produce ethanol was studied, and results were compared to the widely used 2 % Ca-alginate with no dopants, which acted as control. Immobilized cell reactors that were operated using beads doped with 0.25 % polyethylene oxide (PEO) reached an ethanol yield of â¼70 % in 24 h, which was significantly higher than an ethanol yield of 25 % obtained for the control reactor operated using undoped Ca-alginate beads. This study shows that the use of water-soluble dopants can potentially reduce the lag phase and thus improve the initial production yield of immobilized cell reactors, likely due to an increase in porosity and diffusion rate of the doped beads.
Subject(s)
Bioreactors , Cells, Immobilized/chemistry , Ethanol/chemical synthesis , Zymomonas/chemistry , Alginates/chemistry , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Polyethylene Glycols/chemistry , Water/chemistryABSTRACT
Polysaccharide levan is a homopolymer of fructose and is an important component of plants, yeast, fungi and some bacterial biofilms. In this paper we report on the structural properties of aqueous solutions of bacterial levan utilizing smallangle X-ray scattering and light microscopy. In addition to commercially available levan isolated from Zymomonas mobilis and Erwinia herbicola, we also studied levan isolated and purified from the biofilm of Bacillus subtilis. The smallangle X-ray scattering data were analyzed by the string-of-beads model that revealed qualitative differences in the structure of levan molecules. Levan can be represented as a semi-flexible chain that interacts intra- and inter-molecularly and therefore forms various suprastructures on larger size scales. Increasing the concentration of levan makes the levan structure more compact, which was observed on the nano as well as on the micro scale. The structures with most homogeneously distributed polymer local density were found in B. subtilis levan solutions.
Subject(s)
Erwinia/chemistry , Fructans/chemistry , Models, Molecular , Scattering, Small Angle , Water/chemistry , X-Ray Diffraction , Zymomonas/chemistry , Carbohydrate Conformation , SolutionsABSTRACT
BACKGROUND/AIMS: This work is a study of the ability of three recombinant Zymomonas mobilis strains to release ice nucleators into their growth medium. METHODS: The recombinant ice(+)Z. mobilis cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. RESULTS: Cell-free ice nucleators were obtained from all the recombinant ice(+)Z. mobilis cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 µm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. CONCLUSION: These findings indicate that the recombinant ice(+)Z. mobilis cells could be valuable for future use as a source of active cell-free ice nucleation protein.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Zymomonas/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Ice/analysis , Temperature , Zymomonas/chemistry , Zymomonas/genetics , Zymomonas/growth & developmentABSTRACT
Shigella bacteria constitute the causative agent of bacillary dysentery, an acute inflammatory disease causing the death of more than one million humans per year. A null mutation in the tgt gene encoding the tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) was found to drastically decrease the pathogenicity of Shigella bacteria, suggesting the use of Tgt as putative target for selective antibiotics. The enzyme is only functionally active as a homodimer; thus, interference with the formation of its protein-protein interface is an attractive opportunity for therapeutic intervention. To better understand the driving forces responsible for the assembly, stability, and formation of the homodimer, we studied the properties of the residues that establish the dimer interface in detail. We performed site-directed mutagenesis and controlled shifts in the monomer/dimer equilibrium ratio in solution in a concentration-dependent manner by native mass spectrometry and used crystal structure analysis to elucidate the geometrical modulations resulting from mutational variations. The wild-type enzyme exhibits nearly exclusive dimer geometry. A patch of four aromatic amino acids, embedded into a ring of hydrophobic residues and further stabilized by a network of H-bonds, is essential for the stability of the dimer's contact. Accordingly, any perturbance in the constitution of this aromatic patch by nonaromatic residues reduces dimer stability significantly, with some of these exchanges resulting in a nearly exclusively monomeric state. Apart from the aromatic hot spot, the interface comprises an extended loop-helix motif that exhibits remarkable flexibility. In the destabilized mutated variants, the loop-helix motif adopts deviating conformations in the interface region, and a number of water molecules, penetrating into the interface, are observed.
Subject(s)
Pentosyltransferases/chemistry , Protein Multimerization , Zymomonas/enzymology , Dysentery, Bacillary/microbiology , Humans , Models, Molecular , Mutagenesis, Site-Directed , Pentosyltransferases/genetics , Point Mutation , Protein Stability , Shigella/chemistry , Shigella/enzymology , Shigella/genetics , Zymomonas/chemistry , Zymomonas/geneticsABSTRACT
BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (K M 0.06 mM at pH 5), high catalytic efficiencies (1.3 ⢠10(6) M(-1) ⢠s(-1) at pH 5), pHopt of 5.5 and Topt at 45°C. The enzyme is not thermostable (T½ of 18 minutes at 60°C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 Å for Cα when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.
Subject(s)
Amino Acids/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Gluconacetobacter/enzymology , Pyruvate Decarboxylase/chemistry , Pyruvate Decarboxylase/metabolism , Crystallography, X-Ray , Gluconacetobacter/chemistry , Models, Molecular , Phylogeny , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Tertiary , Sarcina/chemistry , Sarcina/enzymology , Sequence Homology, Amino Acid , Substrate Specificity , Zymomonas/chemistry , Zymomonas/enzymologyABSTRACT
The polysaccharide levan is a homopolymer of fructose and appears in nature as an important structural component of some bacterial biofilms. This paper reports the structural and dynamic properties of aqueous solutions of levan of various origin obtained from dynamic rheological, small-angle X-ray scattering, static and dynamic light scattering, as well as density and sound velocity measurements, determination of polymer branching after per-O-methylation, and microscopy. Besides samples of commercially available levan from Zymomonas mobilis and Erwinia herbicola, we also isolated, purified, and studied a levan sample from the biofilm of Bacillus subtilis. The results of dynamic rheological and light scattering measurements revealed very interesting viscoelastic properties of levan solutions even at very low polymer concentrations. The findings were complemented by small-angle X-ray scattering data that revealed some important differences in the structure of the aqueous levan solutions at the molecular level. Besides presenting detailed dynamic and structural results on the polysaccharide systems of various levans, one of the essential goals of this work was to point out the level of structural information that may be obtained for such polymer systems by combining basic physicochemical, rheological, and various light scattering techniques.
Subject(s)
Bacillus subtilis/chemistry , Erwinia/chemistry , Fructans/chemistry , Polysaccharides, Bacterial/chemistry , Zymomonas/chemistry , Biofilms , Carbohydrate Conformation , Fructans/isolation & purification , Light , Rheology , Scattering, Radiation , Solutions , Water/chemistryABSTRACT
The purpose of this study was to assess the stability of the polyfructan levan under different pH solution conditions by monitoring changes in the levan physicochemical properties, such as molar mass (M), root mean square radius (r(rms)), hydrodynamic radius (r(h)), structure factor (r(rms)/r(h)), and aggregation state with respect to solution pH and hydrolysis time. A commercial levan produced from Z. Mobilis was characterized using asymmetric flow field-flow fractionation (AF4) in combination with online multiangle light scattering (MALS) and differential refractive index (dRI) detection. Under neutral pH solution conditions the levan was found to have a M ranging from 10(5) to 5 × 10(7) g/mol, a r(rms) ranging from ~25 to 100 nm and a r(h) from ~3 to 151 nm. Two populations were observed in the sample. One population with a M less than 106 g/mol which represented ~60 % of the sample and a second population with an ultrahigh M up to 5 × 10(7) g/mol, which comprised ~40 % of the sample. The measured r(rms)/r(h) structure factor decreased from 1.8 to 0.65 across the AF4 fractogram indicating that early eluting low M levan species had a random coil configuration and late eluting high M species had more homogeneous spherical structures. The measured apparent density values decreased from 80 to 10 kg/m(3) across the elution profile and suggest that the observed second population also contains aggregates. The stability of levan in different pH conditions ranging from 1.3 to 8.5 was assessed by tracking changes in the average M and r(h), and monitoring the formation of fructose over 1 week. The onset of levan acid hydrolysis was observed to occur sooner at lower pH conditions and no hydrolysis was observed for pH 5.5 and higher.
Subject(s)
Antineoplastic Agents/chemistry , Fractionation, Field Flow/methods , Fructans/chemistry , Polysaccharides, Bacterial/chemistry , Zymomonas/chemistry , Antineoplastic Agents/isolation & purification , Fructans/isolation & purification , Hydrogen-Ion Concentration , Hydrolysis , Light , Molecular Weight , Polysaccharides, Bacterial/isolation & purification , Scattering, RadiationABSTRACT
Apart from competitive active-site inhibition of protein function, perturbance of protein-protein interactions by small molecules in oligodomain enzymes opens new perspectives for innovative therapeutics. tRNA-guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode. A special feature of this enzyme is the short distance between active site and rim of the dimer interface. This suggests design of expanded active-site inhibitors decorated with rigid, needle-type substituents to spike into potential hot spots of the interaction interface. Ligands with attached ethinyl-type substituents have been synthesized and characterized by Kd measurements, crystallography, noncovalent mass spectrometry, and computer simulations. In contrast to previously determined crystal structures with nonextended active-site inhibitors, a well-defined loop-helix motif, involved in several contacts across the dimer interface, falls apart and suggests enhanced flexibility once the spiking ligands are bound. Mass spectrometry indicates significant destabilization but not full disruption of the complexed TGT homodimer in solution. As directed interactions of the loop-helix motif obviously do not determine dimer stability, a structurally conserved hydrophobic patch composed of several aromatic amino acids is suggested as interaction hot spot. The residues of this patch reside on a structurally highly conserved helix-turn-helix motif, which remains unaffected by the bound spiking ligands. Nevertheless, it is shielded from solvent access by the loop-helix motif that becomes perturbed upon binding of the spiking ligands, which serves as a possible explanation for reduced interface stability.