RESUMEN
Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth.
RESUMEN
ß-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 ß-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (ß/α)8 TIM-barrel-like domain, an (α/ß)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. KEY POINTS: ⢠Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively ⢠Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX ⢠The W86A and W86S mutations in AnBX increased the preference for glucoside substrates.
Asunto(s)
Aspergillus niger , Xilosidasas , Aspergillus niger/metabolismo , Cinética , Aminoácidos , Dominio Catalítico , Xilosidasas/metabolismo , Catálisis , Glucósidos , Disulfuros , Especificidad por Sustrato , Glicósido Hidrolasas/metabolismoRESUMEN
Monascus fermented rice, also known as red yeast rice, exhibits a broad spectrum of biological activities due to its chemical constituents, such as monacolins and azaphilone pigments. Here, we cultured Monascus kaoliang KB9 in a liquid malt medium instead of on rice as a carbon source. Eleven known compounds (1-11) containing azaphilones and their early intermediate were isolated and identified. However, this was the first time that angular tricyclic azaphilones, monasfluols A (4) and B (7), acetyl-monasfluol A (5) and monasfluore A (6), were isolated from this species. Interestingly, all isolated tricyclic azaphilones existed exclusively in enol form in CD3OD, as evidenced by NMR spectroscopy. The absolute configuration of compounds 4-7 was also first experimentally identified based on ECD spectroscopy combined with conformational analyses using computational techniques. The assigned stereochemistry of Monascus azaphilones in this work provides essential structural information that will benefit future biological and pharmaceutical investigations.
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Monascus , Monascus/química , Solventes , Benzopiranos/farmacología , Benzopiranos/química , Pigmentos Biológicos/químicaRESUMEN
Two orange pigments, rubropunctatin (1) and monascorubrin (2), along with the yellow pigments, monascin (3) and ankaflavin (4), were isolated from M. kaoliang KB9-fermented rice, also known as red yeast rice. The orange pigments exhibit a broad spectrum of biological activities and appeared to be the major components of this fermented rice. In this work, quantitative 1H NMR (qHNMR) and 13C NMR experiments were used to determine the amounts of the two orange pigments in a crude extract in which most of the 1H NMR signals of the two compounds were indistinguishable. The quantitative values obtained by NMR techniques were found to be similar to those obtained by HPLC. Thus, the combined qHNMR with 13C experiment described in this work could be further developed to quantifying Monascus pigments or other invaluable natural products when qHNMR alone is insufficient for quantitative analysis.
Asunto(s)
Monascus , Pigmentos Biológicos , Cromatografía Líquida de Alta Presión , Fermentación , Espectroscopía de Resonancia Magnética , Monascus/química , Pigmentos Biológicos/químicaRESUMEN
Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real-time quantitative polymerase chain reaction (RT-qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N-(5'-phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid. This accumulation could in turn result in down-regulation of downstream tryptophan biosynthesis genes. Anthranilic acid is typically produced by chemical synthesis and has been used as a substrate for synthesising bioactive compounds including commercial drugs; our results could provide a new biological platform for production of this compound.
Asunto(s)
Saccharomyces cerevisiae , Triptófano , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triptófano/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , ortoaminobenzoatos/farmacología , ortoaminobenzoatos/metabolismoRESUMEN
Maillard reaction products (MRPs) between chitosan and various sugars with enhanced antioxidant activity were previously produced. However, few reports address the chitosan and corn syrup solids system that has been successfully used to encapsulate nutraceutical oils. Maillard reaction is pH-responsive, the influence of solution pH on the formation kinetic and antioxidant activity of MRPs was therefore evaluated in this work. FT-IR and zeta-potential results confirmed the formation of MRPs between chitosan and corn syrup solids. Possible Amadori compounds signals were observed clearly in the 1H NMR spectrum. Brown color development depended on initial solution pH, following a zero-order kinetic regression. Antioxidant activity of reaction products was higher than the native system and increased with an increase in the initial pH of the solution. Developed MRPs with a dual function as antioxidant and encapsulant can possibly be used to protect emulsified oil from oxidation.
Asunto(s)
Antioxidantes , Quitosano , Antioxidantes/química , Quitosano/química , Productos Finales de Glicación Avanzada/química , Concentración de Iones de Hidrógeno , Reacción de Maillard , Espectroscopía Infrarroja por Transformada de Fourier , Zea maysRESUMEN
Alternapyrone is a highly methylated polyene α-pyrone biosynthesised by a highly reducing polyketide synthase. Mutations of the catalytic dyad residues, H1578A/Q and E1604A, of the C-methyltransferase domain resulted in either significantly reduced or no production of alternapyrone, indicating the importance of C-methylation for alternapyrone biosynthesis.
Asunto(s)
Sintasas Poliquetidas , Triterpenos , Metilación , Polienos/química , Sintasas Poliquetidas/metabolismoRESUMEN
Sattahipmycin was isolated from the mycelium of marine-derived Streptomyces sp. GKU 257-1 by following the antibiofilm activity against E. coli NBRC 3972 throughout the purification steps. The structure of sattahipmycin was determined to be a new polycyclic xanthone related to xantholipin but lacking a dioxymethylene and a chlorinated carbon. This compound showed activity toward Gram-positive bacteria and Plasmodium falciparum, antibiofilm formation of Escherichia coli, and cytotoxicity to human cancer cell lines. Using genome sequence data, a biosynthetic pathway leading to sattahipmycin has been proposed involving an uncharacterized type II polyketide synthase biosynthetic gene cluster.
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Streptomyces , Xantonas , Escherichia coli/genética , Bacterias Grampositivas , Humanos , Familia de Multigenes , Streptomyces/química , Xantonas/químicaRESUMEN
The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein-protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Modelos Moleculares , Simulación de Dinámica Molecular , Serratia/química , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Prodigiosina/biosíntesis , Prodigiosina/química , Dominios Proteicos , Serratia/metabolismoRESUMEN
Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and nonreducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP2 and ACP1 -ACP2 )-thioesterase (TE) domains from NR-PKS involved in menisporopsin A biosynthesis. Small-angle X-ray scattering (SAXS) studies in combination with homology modelling reveal that these polypeptides adopt a distinctive beads-on-a-string configuration, characterized by the presence of highly flexible interdomain linkers. These models provide a platform for studying domain organization and interdomain interactions in fungal NR-PKSs, which may be of value in directing the design of functionally optimized polyketide scaffolds.
Asunto(s)
Proteína Transportadora de Acilo/química , Hongos/enzimología , Sintasas Poliquetidas/química , Tioléster Hidrolasas/química , Dicroismo Circular , Macrólidos/química , Simulación de Dinámica Molecular , Dominios Proteicos , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Long-chain alkyl glucosides, such as octyl and decyl ß-d-glucopyranosides (OG and DG, respectively), are regarded as a new generation of biodegradable, non-ionic surfactants. Previously, the mutants of Dalbergia cochinchinensis Pierre dalcochinase showed potential in the synthesis of oligosaccharides and alkyl glucosides. In this study, the N189F dalcochinase mutant gave the highest yields of OG and DG synthesis under reverse hydrolysis conditions. The optimized yield of OG (57.5â¯mol%) was obtained in the reactions containing 0.25â¯M glucose and 0.3 units of the N189â¯F mutant in buffer-saturated octanol at 30⯰C. The identity of OG and DG products was confirmed by high resolution mass spectrometry (HRMS) and NMR. Consistent with its capability for synthesis, the reactivation kinetics and ITC analysis revealed that the aglycone binding pocket of the N189F mutant was more favorable for long-chain alkyl alcohols than the wild-type dalcochinase, while their glycone binding pockets showed similar affinity for the glucosyl moiety. STD NMR revealed higher interactions at the aglycone sites than the glycone sites. Our results demonstrated a promising potential of the N189F dalcochinase mutant in the future commercial production of long-chain alkyl glucosides via reverse hydrolysis reactions.
Asunto(s)
Glucósidos/metabolismo , beta-Glucosidasa/metabolismo , Alcoholes/química , Alcoholes/metabolismo , Catálisis , Dalbergia/enzimología , Glucosa/metabolismo , Glucósidos/química , Cinética , Modelos Moleculares , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , beta-Glucosidasa/química , beta-Glucosidasa/genéticaRESUMEN
OBJECTIVE: To synthesize octyl ß-D-glucopyranoside (OG) and decyl ß-D-glucopyranoside (DG) in three non-aqueous reaction systems, namely organic solvents, ionic liquids and co-solvent mixtures, via reverse hydrolysis reactions catalyzed by the N189F dalcochinase mutant. RESULTS: The highest yield of OG (67 mol%) was obtained in the reaction containing 0.5 M glucose, 3 unit ml-1 enzyme in 20% (v/v) octanol and 70% (v/v) [BMIm][PF6] at 30 °C. On the other hand, the highest yield of DG (64 mol%) was obtained in the reaction containing 0.5 M glucose, 3 unit ml-1 enzyme in 20% (v/v) decanol, 20% (v/v) acetone and 50% (v/v) [BMIm][PF6] at 30 °C. The identities of OG and DG products were confirmed by HRMS and NMR. CONCLUSION: This is the first report of enzymatic synthesis of OG and DG via reverse hydrolysis reactions in ionic liquids and co-solvent mixtures. The N189F dalcochinase mutant and the non-aqueous reaction systems described here show great potential for future commercial production of long-chain alkyl glucosides.
Asunto(s)
Galactósidos/química , Solventes/química , beta-Glucosidasa/metabolismo , Hidrólisis , Líquidos Iónicos/química , Ingeniería de ProteínasRESUMEN
Whole genome analysis of Streptomyces sp. KO-7888 has revealed various pathway-specific transcriptional regulatory genes associated with silent biosynthetic gene clusters. A Streptomyces antibiotic regulatory protein gene, speR, located adjacent to a novel nonribosomal peptide synthetase (NRPS) gene cluster, was overexpressed in the wild-type strain. The resulting recombinant strain of Streptomyces sp. KO-7888 produced two new lipopeptides, sarpeptins A and B. Their structures were elucidated by high-resolution electrospray ionization mass spectrometry, NMR analysis, and the advanced Marfey's method. The distinct modular sections of the corresponding NRPS biosynthetic gene cluster were characterized, and the assembly line for production of the lipopeptide chain was proposed.
Asunto(s)
Lipopéptidos/aislamiento & purificación , Péptido Sintasas/metabolismo , Streptomyces/metabolismo , Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Hongos/efectos de los fármacos , Genes Bacterianos , Genes Reguladores , Lipopéptidos/biosíntesis , Lipopéptidos/química , Lipopéptidos/farmacología , Pruebas de Sensibilidad Microbiana , Conformación Proteica , Streptomyces/genéticaRESUMEN
Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M. theobromae identified an R-PKS gene, men1, and an NR-PKS gene, men2, which both exhibited highest expression levels during the menisporopsin A production phase. These were cloned into separate vectors for heterologous expression in Aspergillus oryzae NSAR1. Unexpectedly, coexpression of the two PKSs alone was sufficient to catalyze the formation of the macrocyclic polylactone, ascotrichalactone A, a structural derivative of menisporopsin A. The unanticipated esterification and cyclolactonization activities could reside in the unusual thioesterase domain of the NR-PKS, which is similar to that of the NRPS catalyzing elongation and cyclization of trilactone in enterobactin biosynthesis and that of modular PKSs catalyzing macrodiolide formation in elaiophylin and conglobatin biosyntheses.
Asunto(s)
Ascomicetos/metabolismo , Aspergillus oryzae/metabolismo , Vías Biosintéticas , Proteínas Fúngicas/metabolismo , Macrólidos/metabolismo , Sintasas Poliquetidas/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Clonación Molecular , Proteínas Fúngicas/genética , Expresión Génica , Genes Fúngicos , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Sintasas Poliquetidas/genética , TranscriptomaRESUMEN
A novel ß-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting ß-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) ß-glucosidase. When expressed in Komagataella pastoris, the recombinant MaBG appeared as a ~ 55-kDa protein without glycosylation modifications. Kinetic parameters as well as the lack of secretion signal suggested that MaBG is an intracellular enzyme and not involved in cellulolysis. The hydrolytic activities of MaBG were enhanced in the presence of up to 3.5-4.5 M glucose, partly due to its strong transglucosylation activity, which suggests its applicability in biosynthetic processes. The potential synthetic activities of the recombinant MaBG were demonstrated in the synthesis of para-nitrophenyl-ß-D-gentiobioside via transglucosylation and octyl glucoside via reverse hydrolysis. The information obtained from this study has broadened our insight into the functional characteristics of this variant of termite GH1 ß-glucosidase and its applications in bioconversion and biotechnology.
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Proteínas de Insectos/química , Isópteros/enzimología , beta-Glucosidasa/química , Animales , Clonación Molecular , Hidrólisis , Proteínas de Insectos/genética , Isópteros/genética , Cinética , Especificidad por Sustrato , beta-Glucosidasa/genéticaRESUMEN
Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-ß-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a subclass ("DpsC type") of priming ketosynthases (KS III). To date, however, no structural information exists for this subclass of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short acyl-CoAs. We present five crystal structures of DpsC, including apo-DpsC, acetyl-DpsC, propionyl-DpsC, butyryl-DpsC, and a cocrystal of DpsC with a nonhydrolyzable phosphopantetheine (PPant) analogue. The DpsC crystal structures reveal the architecture of the active site, the molecular determinants for catalytic activity and homology to O-malonyl transferases, but also indicate distinct differences. These results provide a structural basis for rational engineering of starter unit selection in type II polyketide synthases.
Asunto(s)
Daunorrubicina/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acilación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Malonil Coenzima A/química , Malonil Coenzima A/metabolismo , Modelos Moleculares , Sintasas Poliquetidas/genética , Conformación Proteica , Streptomyces/enzimologíaRESUMEN
Two bioactive cyclic dipeptides, cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr), were isolated from the culture broth of Streptomyces sp. strain 22-4 and tested against three economically important plant pathogens, Xanthomonas axonopodis pv. citri, Ralstonia solanacearum and Clavibacter michiganensis. Both cyclic dipeptides were active against X. axonopodis pv. citri and R. Solanacearum with MIC of 31.25 µg/mL. No activity could be observed against C. michiganensis.
Asunto(s)
Antibacterianos/farmacología , Dipéptidos/farmacología , Péptidos Cíclicos/farmacología , Enfermedades de las Plantas/microbiología , Streptomyces/química , Antibacterianos/aislamiento & purificación , Dipéptidos/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/aislamiento & purificación , Piperazinas/aislamiento & purificación , Piperazinas/farmacología , Ralstonia/efectos de los fármacos , Xanthomonas/efectos de los fármacosRESUMEN
ß-Xylosidases catalyze the breakdown of ß-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 ß-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the ß-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 ß-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry.
Asunto(s)
Aspergillus niger/enzimología , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/metabolismo , Xilosidasas/metabolismo , Clonación Molecular , Codón/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Hidrólisis , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Xilanos , Xilosidasas/química , Xilosidasas/genética , Xilosidasas/aislamiento & purificaciónRESUMEN
Plasmepsin V (PMV) is a Plasmodium aspartic protease responsible for the cleavage of the Plasmodium export element (PEXEL) motif, which is an essential step for export of PEXEL containing proteins and crucial for parasite viability. Here we describe the genetic polymorphism of Plasmodium vivax PMV (PvPMV) Thailand isolates, followed by cloning, expression, purification and characterization of PvPMV-Thai, presenting the pro- and mature-form of PvPMV-Thai. With our refolding and purification method, approximately 1mg of PvPMV-Thai was obtained from 1g of washed inclusion bodies. Unlike PvPMV-Ind and PvPMV-Sal-1, PvPMV-Thai contains a four-amino acid insertion (SVSE) at residues 246-249. We have confirmed that this insertion did not interfere with the catalytic activity as it is located in the long loop (R241-E272) pointing away from the substrate-binding pocket. PvPMV-Thai exhibited similar activity to PfPMV counterparts in which PfEMP2 could be hydrolyzed more efficiently than HRPII. Substrate specificity studies at P1' showed that replacing Ser by Val or Glu of the PfEMP2 peptide markedly reduced the enzyme activity of PvPMV similar to that of PfPMV whereas replacing His by Val or Ser of the HRPII peptide increased the cleavage activity. However, the substitution of amino acids at the P2 position with Glu dramatically reduced the cleavage efficiency by 80% in PvPMV in contrast to 30% in PfPMV, indicating subtle differences around the S2 binding pocket of both PfPMV and PvPMV. Four inhibitors were also evaluated for PvPMV-Thai activity including PMSF, pepstatin A, nelfinavir, and menisporopsin A-a macrocyclic polylactone. We are the first to show that menisporopsin A partially inhibits the PvPMV-Thai activity at high concentration. Taken together, these findings provide insights into recombinant production, substrate specificity and inhibition of PvPMV-Thai.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Malaria Vivax/parasitología , Plasmodium vivax/enzimología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencias de Aminoácidos , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Estabilidad de Enzimas , Eritrocitos/parasitología , Humanos , Cinética , Plasmodium vivax/química , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Especificidad por Sustrato , TailandiaRESUMEN
Type I polyketide synthases often use programmed ß-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where ß-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with ß-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem ß-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting ß-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.