RESUMEN
Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed.
Asunto(s)
Aldehído-Liasas , Escherichia coli , Fructosa-Bifosfato Aldolasa , Simulación del Acoplamiento Molecular , Nucleósidos de Pirimidina , Thermotoga maritima , Animales , Escherichia coli/enzimología , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/síntesis química , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Conejos , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Thermotoga maritima/enzimología , Dihidroxiacetona Fosfato/metabolismo , Dihidroxiacetona Fosfato/química , EstereoisomerismoRESUMEN
Fructose bisphosphate aldolases (FBAs) catalyze the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. We analyzed two previously uncharacterized cytosolic Arabidopsis FBAs, AtFBA4 and AtFBA5. Based on a recent report, we examined the interaction of AtFBA4 with calmodulin (CaM)-like protein 11 (AtCML11). AtFBA4 did not bind AtCML11; however, we found that CaM bound AtFBA5 in a Ca2+-dependent manner with high specificity and affinity (KD ~ 190 nm) and enhanced its stability. AtFBA4 and AtFBA5 exhibited Michaelis-Menten kinetics with Km and Vmax values of 180 µm and 4.9 U·mg-1 for AtFBA4, and 6.0 µm and 0.30 U·mg-1 for AtFBA5, respectively. The flavonoid morin inhibited both isozymes. Our study suggests that Ca2+ signaling and flavanols may influence plant glycolysis/gluconeogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Calmodulina , Flavonoides , Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Calmodulina/metabolismo , Calmodulina/química , Flavonoides/metabolismo , Flavonoides/farmacología , Flavonoides/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/química , Fructosa-Bifosfato Aldolasa/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Calcio/metabolismo , Cinética , Unión Proteica , FlavonasRESUMEN
Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product dihydroxyacetone phosphate reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate-binding pocket, two metal-binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal-dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfoglycolytic Embden-Meyerhof-Parnas gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases have mututally exclusive occurrence within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.
Asunto(s)
Aldehído-Liasas , Fructosa-Bifosfato Aldolasa , Aldehído-Liasas/química , Carbono , Fructosa-Bifosfato Aldolasa/química , Metales , FosfatosRESUMEN
In the present study, the structure of the fructose-1,6-bisphosphataldolase (FBA) gene in Mytilus galloprovincialis (Lamarck, 1819) was analyzed and its tissue specificity of expression level and activity was determined. A 1092 base pairs (bps) complete coding sequence of the FBA gene was assembled from M. galloprovincialis transcriptome. Only one gene encoding FBA (MgFBA) was identified in the M. galloprovincialis genome. The length of MgFBA was 363 amino acids with a molecular mass of 39.7 kDa. According to the amino acid residues, the detected MgFBA gene is a type I aldolase. The FBA gene in M. galloprovincialis had 7 exons; the maximum intron length was about 2.5 kbps. Intraspecific nucleotide diversity (15 mutations) between MgFBAs from the Mediterranean mussels and the Black Sea mussels (present study) was detected. All mutations were synonymous. Tissue specificity in FBA expression level and activity was established. No direct correlation between these functions was found. The highest level of FBA gene expression is found in muscle tissue. According to the phylogenetic analyses, FBA gene in invertebrates could be considered the ancestral gene of muscle type aldolase, which may explain the character of tissue-specific expression.
Asunto(s)
Fructosa-Bifosfato Aldolasa , Mytilus , Mytilus/enzimología , Mytilus/genética , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/genética , Expresión Génica , Evolución Molecular , Filogenia , Secuencia de Aminoácidos , Alineación de Secuencia , Especificidad de ÓrganosRESUMEN
Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.
Asunto(s)
Aldehído-Liasas , Compuestos Epoxi , Compuestos Epoxi/química , Biomasa , Biocatálisis , Aldehído-Liasas/química , Fructosa-Bifosfato Aldolasa/químicaRESUMEN
Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent "Schiff base" intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars.
Asunto(s)
Fructosa-Bifosfato Aldolasa , Transaldolasa , Transaldolasa/química , Transaldolasa/metabolismo , Fructosa-Bifosfato Aldolasa/química , Metilglucósidos/química , Metilglucósidos/metabolismoRESUMEN
Thermostability is important for the thermoactivity of proteins including enzymes. However, it is still challenging to pinpoint the specific structural factors for different temperature thresholds to initiate their specific structural and functional perturbations. Here, graph theory was used to investigate how the temperature-dependent noncovalent interactions as identified in the structures of aldolase B and its prevalent A149P mutant could form a systematic fluidic grid-like mesh network with topological grids to regulate the structural thermostability and the functional thermoactivity upon cyclization against decyclization in an extended range of a subunit. The results showed that the biggest grid may determine the melting temperature thresholds for the changes in their secondary and tertiary structures and specific catalytic activities. Further, a highly conserved thermostable grid may serve as an anchor to secure the flexible active site to achieve the specific thermoactivity. Finally, higher grid-based systematic thermal instability may disfavor the thermoactivity. Thus, this computational study may provide critical clues for the structural thermostability and the functional thermoactivity of proteins including enzymes.
Asunto(s)
Fructosa-Bifosfato Aldolasa , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Temperatura , Estabilidad de EnzimasRESUMEN
Fuculose phosphate aldolases play an important role in glycolysis and gluconeogenesis pathways. L-fuculose 1-phosphate aldolase catalyzes the reversible cleavage of L-fuculose 1-phosphate to DHAP and L-lactaldehyde. Class II aldolases found in bacteria are linked to pathogenesis of human pathogens, and have potential applications in the biosynthesis of carbohydrates and other chiral compounds. Here we report the structure of a putative L-fuculose 1-phosphate aldolase (KpFucA) from the nosocomial pathogen Klebsiella pneumoniae to 1.85 Å resolution. The enzyme crystallizes in space group P422 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis confirms that KpFucA is a tetramer in solution. A magnesium ion cofactor and sulfate ion were identified in the active pocket. Enzyme activity assays confirmed that KpFcuA has a strong preference for L-fuculose 1-phosphate as a substrate, but can also catalyze the cleavage of fructose-1,6-bisphosphate and glucose-6-phosphate. This work should provide a starting point for further investigation of the role of KpFucA in K. pneumoniae pathogenesis or in industrial applications.
Asunto(s)
Fructosa-Bifosfato Aldolasa , Klebsiella pneumoniae , Aldehído-Liasas/metabolismo , Catálisis , Fructosa-Bifosfato Aldolasa/química , Klebsiella pneumoniae/metabolismoRESUMEN
An appealing and challenging cage structure along with an unusual biosynthetic pathway prompted us to explore an expeditious bioinspired one-pot total synthesis of nesteretal A. An unconventional strategy was chosen, and a cascade reaction starting from diacetyl was studied. Under organocatalytic conditions mimicking an aldolase, nesteretal A and a related cage analogue were anticipated by in silico metabolization, detected, targeted, and characterized.
Asunto(s)
Quimioinformática , Diacetil , Fructosa-Bifosfato Aldolasa , Diacetil/química , Diacetil/metabolismo , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Conformación Molecular , EstereoisomerismoRESUMEN
Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an â¼2.3â Å resolution reconstruction of mouse apoferritin (â¼500â kDa) and an â¼3.3â Å resolution reconstruction of rabbit muscle aldolase (â¼160â kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.
Asunto(s)
Microscopía por Crioelectrón/métodos , Glicerol/química , Animales , Apoferritinas/química , Tampones (Química) , Fructosa-Bifosfato Aldolasa/química , Ratones , Modelos Moleculares , Pliegue de Proteína , ConejosRESUMEN
Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis, liquid chromatography-mass spectrometry, and the crystallographic structures of APO-CaFBA, CaFBA-G3P, and C157S-2a4 revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant C. albicans, and compound 2a11 can inhibit the growth of azole-resistant strains 103 with the MIC80 of 1 µg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Farmacorresistencia Fúngica/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Proteínas Fúngicas/antagonistas & inhibidores , Sitio Alostérico , Antifúngicos/síntesis química , Antifúngicos/metabolismo , Azoles/síntesis química , Azoles/metabolismo , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Candida parapsilosis/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Some protein positions play special roles in determining the magnitude of protein function: at such "rheostat" positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A ("I98X"). Despite being ~17 Å from the active site and far from subunit interfaces, substitutions at position 98 have rheostatic contributions to the apparent cooperativity (nH ) associated with fructose-1,6-bisphosphate substrate binding and moderately affected binding affinity. Next, we crystallized representative I98X variants to assess structural consequences. Residues smaller than the native isoleucine (cysteine and serine) were readily accommodated, and the larger phenylalanine caused only a slight separation of the two parallel helixes. However, the diffraction quality was reduced for I98F, and further reduced for I98Y. Intriguingly, the resolutions of the I98X structures correlated with their nH values. We propose that substitution effects on both nH and crystal lattice disruption arise from changes in the population of aldolase A conformations in solution. In combination with results computed for rheostat positions in other proteins, the results from this study suggest that rheostat positions accommodate a wide range of side chains and that structural consequences manifest as shifted ensemble populations and/or dynamics changes.
Asunto(s)
Fructosa-Bifosfato Aldolasa , Sustitución de Aminoácidos , Sitios de Unión , Dominio Catalítico , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/genética , Humanos , Mutación Missense , Conformación ProteicaRESUMEN
Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaIâ¢M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaIâ¢Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaIâ¢M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by â¼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaIâ¢Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.
Asunto(s)
Acinetobacter baumannii/enzimología , Calcio/química , Fructosa-Bifosfato Aldolasa/química , Zinc/química , Proteínas Bacterianas , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Especificidad por SustratoRESUMEN
Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data-acquisition approaches in single-particle cryo-EM do not take it into account. Here, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated data collection to images with preserved high-resolution details. This quality-over-quantity approach almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to a lack of high-resolution information. It maximizes the data-collection efficiency and lowers the electron-microscopy time required per data set. This strategy is especially useful if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck.
Asunto(s)
Microscopía por Crioelectrón/métodos , Fructosa-Bifosfato Aldolasa/química , Procesamiento de Imagen Asistido por Computador/métodos , Manejo de Especímenes/métodos , Animales , Conejos , Programas InformáticosRESUMEN
The retro-aldolase mechanism of methodol catalysed by the catalytic antibody 33F12 is described based on the exploration of the free energy landscape obtained with QM/MM methods. The amino acids involved in the reaction have been identified, as well as their specific role played in the active site and in the flexibility of the loops. Finally, the comparison with a de novo enzyme RA95.5-8F provides a deeper understanding of catalytic differences between such different protein scaffolds.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Teoría Cuántica , Anticuerpos Catalíticos/química , Biocatálisis , Cristalografía por Rayos X , Fructosa-Bifosfato Aldolasa/química , Metanol/química , Metanol/metabolismo , Simulación de Dinámica MolecularRESUMEN
N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.
Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Escherichia coli/enzimología , Fructosa-Bifosfato Aldolasa/química , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Periplasma/metabolismo , Conformación Proteica , Transporte de Proteínas , EstereoisomerismoRESUMEN
The 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) enzyme in psychrophilic bacteria has gradually attracted the attention of researchers. A novel gene, deoC (681 bp), encoding DERAPsy, was identified in Pseudomonas syringae pv. syringae B728a, recombinantly expressed in E. coli BL21 and purified via affinity chromatography, which yielded a homodimeric enzyme of 23 kDa. The specific activity of DERAPsy toward 2-deoxy-d-ribose-5-phosphate (DR5P) was 7.37 ± 0.03 U/mg, and 61.32% of its initial activity remained after incubation in 300 mM acetaldehyde at 25 °C for 2 h. Based on the calculation results (dock binding free energy) with the ligand chloroacetaldehyde (CAH), five target substitutions (T16L, F69R, V66K, S188V, and G189R) were identified, in which the DERAPsy mutant (G189R) exhibited higher catalytic activity toward DR5P than DERAPsy. Only the DERAPsy mutant (V66K) exhibited 12% higher activity toward chloroacetaldehyde and acetaldehyde condensation reactions than DERAPsy. Fortunately, the aldehyde tolerance of these mutants exhibited no significant decline compared with the wild type. These results indicate an effective strategy for enhancing DERA activity.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Bacterianas , Fructosa-Bifosfato Aldolasa , Mutación Missense , Pseudomonas syringae , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Catálisis , Fructosa-Bifosfato Aldolasa/biosíntesis , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/aislamiento & purificación , Pseudomonas syringae/enzimología , Pseudomonas syringae/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
BACKGROUND: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). METHODS: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. RESULTS: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer â hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. CONCLUSIONS: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaBâ PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. GENERAL SIGNIFICANCE: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Glucosa-6-Fosfato Isomerasa/metabolismo , Glutamato Descarboxilasa/metabolismo , Pirofosfatasa Inorgánica/metabolismo , Proteínas de la Membrana/metabolismo , Escherichia coli/química , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Fructosa-Bifosfato Aldolasa/química , Glucosa-6-Fosfato Isomerasa/química , Glutamato Descarboxilasa/química , Humanos , Pirofosfatasa Inorgánica/química , Cinética , Proteínas de la Membrana/química , Multimerización de ProteínaRESUMEN
l-2-Keto-3-deoxyarabinonate (l-KDA) dehydratase (AraD) catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde in the nonphosphorylative l-arabinose pathway from bacteria and belongs to the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein superfamily. All members of this superfamily, including several aldolases for l-KDA, share a common catalytic mechanism of retro-aldol fission, in which a lysine residue forms a Schiff base with the carbonyl C2 atom of the substrate, followed by proton abstraction of the substrate by a tyrosine residue as the base catalyst. Only AraD possesses a glutamine residue instead of this active site tyrosine, suggesting its involvement in catalysis. We herein determined the crystal structures of AraD from the nitrogen-fixing bacterium Azospirillum brasilense and AraD in complex with ß-hydroxypyruvate and 2-oxobutyrate, two substrate analogues, at resolutions of 1.9, 1.6, and 2.2 Å, respectively. In both of the complexed structures, the ε-nitrogen of the conserved Lys171 was covalently linked to the carbonyl C2 atom of the ligand, which was consistent with the Schiff base intermediate form, similar to other DHDPS/NAL members. A site-directed mutagenic study revealed that Glu173 and Glu200 played important roles as base catalysts, whereas Gln143 was not absolutely essential. The abstraction of one of the C3 protons of the substrate (but not the O4 hydroxyl) by Glu173 was similar to that by the (conserved) tyrosine residues in the two DHDPS/NAL members that produce α-ketoglutaric semialdehyde (d-5-keto-4-deoxygalactarate dehydratase and Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase), indicating that these enzymes evolved convergently despite similarities in the overall reaction.
Asunto(s)
Biocatálisis , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Azospirillum brasilense/enzimología , Fructosa-Bifosfato Aldolasa/genética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.