Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer.

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

Quality comparison of a state-of-the-art preparation of a recombinant L-asparaginase derived from Escherichia coli with an alternative asparaginase product.

L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylated Escherichia coli (E. coli)-derived ASNase as well as Erwinia chrysanthemi-derived ASNase. Additionally, a new recombinant E. coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinant E. coli-derived ASNase marketed in Europe (Spectrila®) with an E. coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila® also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, the E. coli DNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila® met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited.

Decreasing the immunogenicity of Erwinia chrysanthemi asparaginase via protein engineering: computational approach.

Immunogenicity of therapeutic proteins is one of the main challenges in disease treatment. L-Asparaginase is an important enzyme in cancer treatment which sometimes leads to undesirable side effects such as immunogenic or allergic responses. Here, to decrease Erwinase (Erwinia chrysanthemiL-Asparaginase) immunogenicity, which is the main drawback of the enzyme, firstly conformational B cell epitopes of Erwinase were predicted from three-dimensional structure by three different computational methods. A few residues were defined as candidates for reducing immunogenicity of the protein by point mutation. In addition to immunogenicity and hydrophobicity, stability and binding energy of mutants were also analyzed computationally. In order to evaluate the stability of the best mutant, molecular dynamics simulation was performed. Among mutants, H240A and Q239A presented significant reduction in immunogenicity. In contrast, the immunogenicity scores of D235A slightly decreased according to two servers. Binding affinity of substrate to the active site reduced significantly in K265A and E268A. The final results of molecular dynamics simulation indicated that H240A mutation has not changed the stability, flexibility, and the total structure of desired protein. Overall, point mutation can be used for reducing immunogenicity of therapeutic proteins, in this context, in silico approaches can be used to screen suitable mutants.

L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression.

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

In vitro screening and in silico validation revealed key microbes for higher production of significant therapeutic enzyme l-asparaginase.

l-asparaginase is an enzyme of medical prominence and reputable as a chemotherapeutic agent. It also has immense potential to cure autoimmune and infectious diseases. The vast application of this enzyme in healthcare sector increases its market demand. However, presently the huge market demand is not achieved completely. This serves the basis to explore better producer microbial strains to bridge the gap between huge demand and supply of this therapeutic enzyme. The present study deals with the successful screening of potent microorganisms producing l-asparaginase. 47 microorganisms were screened including bacteria, fungi, and yeasts. Among all, Penicillium lilacinum showed the highest enzyme activity i.e., 39.67 IU/ml. Shigella flexneri has 23.21 IU/ml of enzyme activity (highest among all the bacterial strain tested). Further, the 3-D structure of l-asparaginase from higher producer strains was developed and validated in silico for its activity. l-asparagine (substrate for l-asparaginase) was docked inside the binding pocket of P. lilacinum and S. flexneri. Docking score for the most common substrate l-asparagine is -6.188 (P. lilacinum), -5.576 (S. flexneri) which is quite good. Moreover, the chemical property of the binding pocket revealed that amino acid residues Phe 243, Gln 260, Gly 365, Asp 386 in P. lilacinum and residues Asp 181, Thr 318, Asn 320 in S. flexneri have an important role in H-bonding. The in silico results supports and strengthen the wet lab results. The outcome obtained motivates to take the present study result from lab to industry for the economic/massive production of this enzyme for the diverse therapeutic application.

Attenuation and quantitation of virulence gene expression in quorum-quenched Dickeya chrysanthemi.

N-Acyl-homoserine lactones (AHLs)-dependent quorum sensing (QS) system(s) is recruited by the soft rot bacterium Dickeya chrysanthemi for coordinating its social activities such as secretion of plant cell wall-degrading enzymes, while the main signal molecule and quantity dependence of virulence to QS in this bacterium have not been clarified. To do this end, the involvement of AHLs in African violet leaves and potato tuber maceration; swarming motility; pectate lyase and polygalacturonase enzymes production and in planta expression of virulence genes including pelE, pehX and pemA by electroporating two quorum-quenching vectors. The expression of two types of AHL-lactonase expressing vector caused dramatic decrease in swarming motility, production of pectinolytic enzymes and macerating of plant tissues. The maximum ability of quenching of QS in repression of D. chrysanthemi virulence was assessed quantitatively by q-RT-PCR, as expression of pelE, pehX and pemA genes were decreased 90.5-92.18 % in quenched cells. We also showed that virulence and pathogenicity of this bacterium was under the control of DHL-dependent QS system and that the existence of second DHL operating system is probable for this bacterium. Thus, this signal molecule would be the key point for future research to design DHL-specific lactonase enzymes using bioinformatics methods.

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.

Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects.

Consensus expert recommendations for identification and management of asparaginase hypersensitivity and silent inactivation.

L-asparaginase is an integral component of therapy for acute lymphoblastic leukemia. However, asparaginase-related complications, including the development of hypersensitivity reactions, can limit its use in individual patients. Of considerable concern in the setting of clinical allergy is the development of neutralizing antibodies and associated asparaginase inactivity. Also problematic in the use of asparaginase is the potential for the development of silent inactivation, with the formation of neutralizing antibodies and reduced asparaginase activity in the absence of a clinically evident allergic reaction. Here we present guidelines for the identification and management of clinical hypersensitivity and silent inactivation with Escherichia coli- and Erwinia chrysanthemi- derived asparaginase preparations. These guidelines were developed by a consensus panel of experts following a review of the available published data. We provide a consensus of expert opinions on the role of serum asparaginase level assessment, indications for switching asparaginase preparation, and monitoring after change in asparaginase preparation.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

Chromosomal "stress-response" domains govern the spatiotemporal expression of the bacterial virulence program.

UNLABELLED: Recent studies strongly suggest that the gene expression sustaining both normal and pathogenic bacterial growth is governed by the structural dynamics of the chromosome. However, the mechanistic device coordinating the chromosomal configuration with selective expression of the adaptive traits remains largely unknown. We used a holistic approach exploring the inherent relationships between the physicochemical properties of the DNA and the expression of adaptive traits, including virulence factors, in the pathogen Dickeya dadantii (formerly Erwinia chrysanthemi). In the transcriptomes obtained under adverse conditions encountered during bacterial infection, we explored the patterns of chromosomal DNA sequence organization, supercoil dynamics, and gene expression densities, together with the long-range regulatory impacts of the abundant DNA architectural proteins implicated in pathogenicity control. By integrating these data, we identified transient chromosomal domains of coherent gene expression featuring distinct couplings between DNA thermodynamic stability, supercoil dynamics, and virulence traits. IMPORTANCE: We infer that the organization of transient chromosomal domains serving specific functions acts as a fundamental device for versatile adjustment of the pathogen to environmental stress. We believe that the identification of chromosomal "stress-response" domains harboring distinct virulence traits and mediating the cellular adaptive behavior provides a breakthrough in understanding the control mechanisms of bacterial pathogenicity.

Regulators involved in Dickeya solani virulence, genetic conservation, and functional variability.

Bacteria from the genus Dickeya (formerly Erwinia chrysanthemi) are plant pathogens causing severe diseases in many economically important crops. A majority of the strains responsible for potato disease in Europe belong to a newly identified Dickeya solani species. Although some ecological and epidemiological studies have been carried out, little is known about the regulation of D. solani virulence. The characterization of four D. solani strains indicates significant differences in their virulence on potato, although they are genetically similar based on genomic fingerprinting profiles. A phenotypic examination included an analysis of virulence on potato; growth rate in culture; motility; Fe3+ chelation; and pectate lyase, cellulase, protease, biosurfactant, and blue pigment production. Mutants of four D. solani strains were constructed by inactivating the genes coding either for one of the main negative regulators of D. dadantii virulence (kdgR, pecS, and pecT) or for the synthesis and perception of signaling molecules (expI and expR). Analysis of these mutants indicated that PecS, PecT, and KdgR play a similar role in both species, repressing, to different degrees, the synthesis of virulence factors. The thermoregulator PecT seems to be a major regulator of D. solani virulence. This work also reveals the role of quorum sensing mediated by ExpI and ExpR in D. solani virulence on potato.

Rhizobium etli asparaginase II: an alternative for acute lymphoblastic leukemia (ALL) treatment.

Bacterial L-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant L-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II L-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity.

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals.

The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinic-receptor superfamily.

Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system.

The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC. The periplasmic regions of GspC and GspD consist of several structural domains, HR(GspC) and PDZ(GspC), and N0(GspD) to N3(GspD), respectively, and recent structural and functional studies have proposed several interaction sites between these domains. We used cysteine mutagenesis and disulfide bonding analysis to investigate the organization of GspC and GspD protomers and to map their interaction sites within the secretion machinery of the plant pathogen Dickeya dadantii. At least three distinct GspC-GspD interactions were detected, and they involve two sites in HR(GspC), two in N0(GspD), and one in N2(GspD). None of these interactions occurs through static interfaces because the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites halts secretion, indicating the transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR(GspC) structure. The T2SS components OutE/L/M affect various interaction sites differently, reinforcing some but diminishing the others, suggesting a possible switching mechanism of these interactions during secretion. Disulfide mapping shows that the organization of GspD and GspC subunits within the T2SS could be compatible with a hexamer of dimers arrangement rather than an organization with 12-fold rotational symmetry.

Dickeya dadantii, a plant pathogenic bacterium producing Cyt-like entomotoxins, causes septicemia in the pea aphid Acyrthosiphon pisum.

Dickeya dadantii (syn. Erwinia chrysanthemi) is a plant pathogenic bacteria that harbours a cluster of four horizontally-transferred, insect-specific toxin genes. It was recently shown to be capable of causing an acute infection in the pea aphid Acyrthosiphon pisum (Insecta: Hemiptera). The infection route of the pathogen, and the role and in vivo expression pattern of these toxins, remain unknown. Using bacterial numeration and immunolocalization, we investigated the kinetics and the pattern of infection of this phytopathogenic bacterium within its insect host. We compared infection by the wild-type strain and by the Cyt toxin-deficient mutant. D. dadantii was found to form dense clusters in many luminal parts of the aphid intestinal tract, including the stomach, from which it invaded internal tissues as early as day 1 post-infection. Septicemia occurred soon after, with the fat body being the main infected tissue, together with numerous early infections of the embryonic chains showing embryonic gut and fat body as the target organs. Generalized septicemia led to insect death when the bacterial load reached about 10(8) cfu. Some individual aphids regularly escaped infection, indicating an effective partial immune response to this bacteria. Cyt-defective mutants killed insects more slowly but were capable of localisation in any type of tissue. Cyt toxin expression appeared to be restricted to the digestive tract where it probably assisted in crossing over the first cell barrier and, thus, accelerating bacterial diffusion into the aphid haemocel. Finally, the presence of bacteria on the surface of leaves hosting infected aphids indicated that the insects could be vectors of the bacteria.

PecS is an important player in the regulatory network governing the coordinated expression of virulence genes during the interaction between Dickeya dadantii 3937 and plants.

Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence.

Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass.

Ethanologenic Escherichia coli strain KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) as well as a pectate lyase (pelE) from Erwinia chrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB pelE), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway out genes from E. chrysanthemi were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (ogl) from E. chrysanthemi was added to produce strain JP08C. E. coli strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, ≤6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.

Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase.

UNLABELLED: Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid ß-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronosyl-(1→2)]-ß-D-xylopyranosyl-(1→4)-D-xylose as a ligand. The crystal structure of the enzyme-ligand complex was solved at 1.39 Å resolution. The ligand xylotriose moiety occupies subsites -1, -2 and -3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear ß-1,4-xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. DATABASE: Structural and experimental data are available in the Protein Data Bank database under accession number 2y24 [45].

Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid.

The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.