RESUMO
Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.
Assuntos
Endosperma/enzimologia , Metabolismo Energético/fisiologia , Proteínas de Plantas/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Zea mays/enzimologia , Endosperma/genética , Mutação , Proteínas de Plantas/genética , Piruvato Ortofosfato Diquinase/genética , Amido/biossíntese , Amido/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/genéticaRESUMO
Cereal yields decrease when grain fill proceeds under conditions of prolonged, moderately elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development. This study used controlled environments to isolate temperature as the sole environmental variable during Zea mays kernel-fill, from 12 days after pollination to maturity. Plants subjected to elevated day, elevated night temperatures (38°C day, 28°C night (38/28°C])) or elevated day, normal night (38/17°C), were compared with those from controls grown under normal day and night conditions (28/17°C). Progression of change over time in endosperm tissue was followed to dissect contributions at multiple levels, including transcriptome, metabolome, enzyme activities, product accumulation, and tissue ultrastructure. Integrated analyses indicated that the normal developmental program of endosperm is fully executed under prolonged high-temperature conditions, but at a faster rate. Accelerated development was observed when both day and night temperatures were elevated, but not when daytime temperature alone was increased. Although transcripts for most components of glycolysis and respiration were either upregulated or minimally affected, elevated temperatures decreased abundance of mRNAs related to biosynthesis of starch and storage proteins. Further analysis of 20 central-metabolic enzymes revealed six activities that were reduced under high-temperature conditions, indicating candidate roles in the observed reduction of grain dry weight. Nonetheless, a striking overall resilience of grain filling in the face of elevated temperatures can be attributed to acceleration of normal endosperm development.
Assuntos
Endosperma/metabolismo , Zea mays/metabolismo , Grão Comestível/genética , Grão Comestível/metabolismo , Grão Comestível/fisiologia , Endosperma/genética , Endosperma/fisiologia , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Temperatura , Zea mays/genética , Zea mays/fisiologiaRESUMO
Recent data suggest alternative mechanisms that promote human leukocyte antigen (HLA)-associated drug syndromes. Hypersensitive responses have been attributed to drug interactions with HLA molecules, peptides presented by HLA molecules and T-cell antigen receptors. Definition of an increasing number of HLA-associated drug syndromes suggests that polymorphism in the antigen-binding cleft residues influence recognition of specific drugs. Recent data demonstrate that small molecule drugs bind within the antigen-binding cleft of HLA in a manner that alters the repertoire of HLA-bound peptide ligands. This drug recognition mechanism permits presentation of self-peptides to which the host has not been tolerized. This altered repertoire mechanism is analogous to massive polyclonal T-cell responses occurring in mismatched HLA organ transplantation in which the drug in effect creates a novel HLA allele. Alteration of the self-peptide repertoire by HLA-binding small molecules may be the mechanistic basis for a diverse set of deleterious T-cell responses since the antigen-binding cleft has structural features that are compatible with binding drug-like small molecules. Small molecule drugs that bind elements of the trimolecular complex (T-cell receptor, peptide, and HLA) may cause short- and long-term adverse effects by a diverse set of mechanisms.
Assuntos
Autoantígenos/química , Hipersensibilidade a Drogas/imunologia , Antígenos HLA/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T/química , Xenobióticos/química , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/imunologia , Autoantígenos/metabolismo , Sítios de Ligação , Hipersensibilidade a Drogas/etiologia , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Humanos , Ligantes , Modelos Moleculares , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Síndrome , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Xenobióticos/efeitos adversosRESUMO
The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADP-glucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33 °C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only â¼50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.
Assuntos
Plantas Geneticamente Modificadas/genética , Sementes/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Zea mays/genética , Flores/genética , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , Temperatura , TransgenesRESUMO
Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase). After each round of mutagenesis, iodine staining and antibody capture activity assays at varying temperatures were used to select the optimum positions and amino acid changes for the next rounds of mutagenesis. After three iterations, the signals from whole-colony iodine staining were saturated and a heat stable AGPase variant was obtained. Kinetic studies of the heat stable mutant showed that it also had an unexpected increased affinity for the activator, 3-PGA. This is particularly valuable as both the temperature stability and allosteric properties of AGPase significantly influence grain yield.
Assuntos
Endosperma/enzimologia , Estabilidade Enzimática , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Zea mays/enzimologia , Cristalografia por Raios X , Endosperma/química , Endosperma/genética , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase/química , Temperatura Alta , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Zea mays/química , Zea mays/genéticaRESUMO
Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Osteoporose/tratamento farmacológico , Osteoprotegerina/química , Animais , Artrite Reumatoide/patologia , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/patologia , Humanos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteoporose/patologia , Osteoprotegerina/administração & dosagem , Polímeros/administração & dosagem , Polímeros/química , RatosRESUMO
Threonine aldolases catalyze the pyridoxal phosphate-dependent condensation between small amino acids (principally glycine) and aldehydes such as acetaldehyde. Carbon-carbon bond formation involves forming two adjacent chiral centers. As a rule, threonine aldolases are very stereoselective for α-carbon configuration but show modest selectivity at the ß-carbon. On the other hand, these enzymes accept a wide variety of synthetically useful acceptor aldehydes, making them important additions to the synthetic toolkit. This review briefly summarizes the reaction mechanism and then lists all published synthetic reactions by threonine aldolases as of early 2014. The current state of the art in crystallographic and protein engineering studies of these enzymes is also presented.
Assuntos
Glicina Hidroximetiltransferase/metabolismo , Catálise , Cristalografia , Estabilidade Enzimática , Glicina Hidroximetiltransferase/química , Engenharia de ProteínasRESUMO
An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes. Several substrates were sterically demanding, and as hoped, creating additional active site volume yielded better conversions for these alkenes. The most impressive improvement was found for 2-butylidenecyclohexanone. The wild-type provided less than 20% conversion after 24h; a triple mutant afforded more than 60% conversion in the same time period. Moreover, even wild-type OYE 2.6 can reduce cyclohexenones with very bulky 4-substituents efficiently.
Assuntos
Biocatálise , Mutagênese Sítio-Dirigida , Oxirredutases/genética , Oxirredutases/metabolismo , Pichia/enzimologia , Alcenos/química , Alcenos/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Oxirredutases/químicaRESUMO
ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis.
Assuntos
Glucose-1-Fosfato Adenililtransferase/química , Proteínas de Plantas/química , Regulação Alostérica , Endosperma/enzimologia , Ativação Enzimática , Ativadores de Enzimas/química , Estabilidade Enzimática , Glucose-1-Fosfato Adenililtransferase/genética , Ácidos Glicéricos/química , Temperatura Alta , Cinética , Oxirredução , Fosfatos/química , Proteínas de Plantas/genética , Tubérculos/enzimologia , Subunidades Proteicas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Solanum tuberosum/enzimologia , Zea mays/enzimologiaRESUMO
ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.
Assuntos
Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/classificação , Fosfatos/química , Tubérculos/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Solanum tuberosum/enzimologia , Zea mays/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/química , Estabilidade Enzimática , Solanum tuberosum/genéticaRESUMO
The sustainable extraction of secondary metabolites from Brassica agro-industrial by-products often involves the use of high concentrations of ethanol, and/or high temperatures, which tends to decrease the efficiency of protein extraction (yield, profile, etc.). To understand the limits of the combination of these two extraction processes, aqueous ethanol extraction of secondary metabolites (e.g., phenolic compounds and glucosinolates) from Brassica carinata defatted meal was optimized using Response Surface Methodology. The validated models predicted that aqueous ethanol extraction of defatted Carinata meal, with a low aqueous EtOH concentration (22% EtOH) at moderate Te (50 °C), enables the efficient recovery of secondary metabolites (sinapine = 9.12 ± 0.05 mg/gDM, sinigrin = 86.54 ± 3.18 µmol/gDM) while maintaining good protein extractability (59.8 ± 2.1%) from successive alkaline extractions. The evaluation of functional properties of the resulting protein isolates revealed that aqueous extraction, under optimized conditions, improves foaming activity while preserving emulsion ability.
RESUMO
ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA--with or without 5.0 mm inorganic phosphate--ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K(M) values for both substrates.
Assuntos
Endosperma/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/enzimologia , Difosfatos/metabolismo , Ácidos Glicéricos/metabolismo , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined--in both the small and large subunits--bound 3-PGA more weakly than the wild type (A(50) increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.
Assuntos
Endosperma/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Glucose-1-Fosfato Adenililtransferase/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Subunidades ProteicasRESUMO
Encapsulating drugs within hollow nanotubes offers several advantages, including protection from degradation, the possibility of targeting desired locations, and drug release only under specific conditions. Template synthesis utilizes porous membranes prepared from alumina, polycarbonate, or other materials that can be dissolved under specific conditions. The method allows for great control over the lengths and diameters of nanotubes; moreover, tubes can be constructed from a wide variety of tube materials including proteins, DNA, silica, carbon, and chitosan. A number of capping strategies have been developed to seal payloads within nanotubes. Combining these advances with the ability to target and internalize nanotubes into living cells will allow these assemblies to move into the next phase of development, in vivo experiments.
Assuntos
Óxido de Alumínio/química , Sistemas de Liberação de Medicamentos/métodos , Nanotubos/química , Dióxido de Silício/química , NanotecnologiaRESUMO
Sinapic acid (SinA) and corresponding esters are secondary metabolites abundantly found in plants of Brassica family. Belonging to the family of p-hydroxycinnamic acids, SinA and its esters analogues are present in different plant parts and involved in multiple biological processes in planta. Moreover, these metabolites are also found in relatively large quantities in agro-industrial wastes. Nowadays, these metabolites are increasingly drawing attention due to their bioactivities which include antioxidant, anti-microbial, anti-cancer and UV filtering activities. As a result, these metabolites find applications in pharmaceutical, cosmetic and food industries. In this context, this article reviews innate occurrence, biosynthesis, accessibility via chemical synthesis or direct extraction from agro-industrial wastes. Biological activities of SinA and its main corresponding esters will also be discussed.
RESUMO
Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C16-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's. The resulting library was initially screened for the reaction between l-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378â¯K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA's. These data showed that the R378â¯K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.
Assuntos
Acil Coenzima A/metabolismo , Engenharia Metabólica/métodos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Sphingomonas/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Especificidade por SubstratoRESUMO
Site-saturation mutagenesis was used to generate all possible replacements for Trp 116 of Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis ) old yellow enzyme (OYE). Our original hypothesisthat smaller amino acids at position 116 would allow better acceptance of bulky 3-alkyl-substituted 2-cyclohexenonesproved incorrect. Instead, Phe and Ile replacements favored the binding of some substrates in an opposite orientation, which yielded reversed stereochemical outcomes compared to that of the wild-type OYE. For example, W116I OYE reduced (R)- and (S)-carvone to enantiomeric products, rather than the diastereomers produced by the wild-type OYE. Deuterium labeling revealed that (S)-carvone reduction by the W116I OYE occurred by the same pathway as that by the wild type (net trans-addition of H(2)), proving that different substrate binding orientations were responsible for the divergent products. Trp 116 mutants also afforded different stereochemical outcomes for reductions of (R)-perillaldehyde and neral. Preliminary studies of an OYE family member whose native sequence contains Ile at position 116 ( Pichia stipitis OYE 2.6) revealed that this enzyme's stereoselectivity matched that of the wild-type S. pastorianus OYE, showing that the identity of the residue at position 116 does not solely determine the substrate binding orientation. Computational docking studies using an induced fit methodology successfully reproduced the majority of the experimental outcomes. These computational tools will allow preliminary in silico screening of additional residues to identify those most likely to control the substrate binding orientation and provide some guidance to future experimental studies.
Assuntos
Monoterpenos/química , NADPH Desidrogenase/química , Saccharomyces/enzimologia , Triptofano/química , Substituição de Aminoácidos , Biocatálise , Monoterpenos Cicloexânicos , Modelos Moleculares , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Oxirredução , EstereoisomerismoRESUMO
The substrate selectivity of the Trp416Gly mutant of Methanothermobacter thermautotrophicus acetyl-CoA synthetase (Trp416Gly MT-ACS1) was explored. The goal was to identify its substrate range, particularly for functionalized carboxylic acid substrates that would allow post-synthesis functionalization of CoA thioesters or downstream products using metathesis or Click chemistry. Relative activities were determined by in situ formation of acyl-hydroxamate iron (III) complexes. Trp416Gly MT-ACS1 showed good activities for saturated straight chain carboxylic acids from C2 to C8, for ω-alkenyl straight chain carboxylic acids from C4 to C7 and for ω-alkynyl straight chain carboxylic acids from C5 to C7. Carboxylic acids showing ≥20% conversion in screening reactions were used in preparative conversions that completely consumed the added CoASH.
Assuntos
Acetato-CoA Ligase/metabolismo , Substituição de Aminoácidos , Methanobacteriaceae/enzimologia , Proteínas Mutantes/metabolismo , Acetato-CoA Ligase/genética , Ácidos Carboxílicos/metabolismo , Proteínas Mutantes/genética , Mutação Puntual , Especificidade por SubstratoRESUMO
Rat NADP-dependent leukotriene B4 12-hydroxydehydrogenase (Ltb4dh) catalyzes olefin reductions for some activated alkenes at the expense of NADPH in the absence of a flavin cofactor. Unlike flavoprotein alkene reductases, where net trans-addition of hydrogen has been consistently observed, Ltb4dh reduced both enantiomers of perillaldehyde to the same cis-product. To uncover the reason for this unexpected result, the stereochemical courses of perillaldehyde reductions by Ltb4dh were determined by deuterium labeling followed by (2)H NMR analysis. These data showed unequivocally that Ltb4dh mediated net trans-addition of hydrogen to (R)-perillaldehyde but followed the opposite stereochemical course (net syn-addition) for (S)-perillaldehyde. To the best of our knowledge, such divergent stereochemical pathways for a single enzyme have not previously been reported.
Assuntos
Oxirredutases do Álcool/química , Alcenos/química , Hidrogênio/química , Monoterpenos/química , Oxirredutases do Álcool/genética , Animais , Catálise , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Ratos , Estereoisomerismo , Especificidade por SubstratoRESUMO
Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia coli (Aeromonas jandaeil-allo-threonine aldolase, E. colil-threonine aldolase and Thermotoga maritimal-allo-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme. These conditions were used to characterize the substrate- and stereoselectivity of each TA toward a panel of aldehyde acceptors. In general, the A. jandaei TA performed best, and six representative conversions were scaled up 10-fold in order to develop downstream steps for product isolation. One key improvement was to treat the crude reaction product with Bacillus subtilis glycine oxidase, which eliminated residual starting material and significantly simplified product isolation. NMR studies were used to identify the major and minor diastereomers from the preparative-scale reactions and the absolute configurations for three representative cases.