CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Plants Utilize a Protection/Deprotection Strategy in Limonoid Biosynthesis: A "Missing Link" Carboxylesterase Boosts Yields and Provides Insights into Furan Formation.

The furan ring is a defining feature of limonoids, a class of highly rearranged and bioactive plant tetranortriterpenoids. We recently reported an apparent complete biosynthetic pathway to these important natural furanoids. Herein, we disclose the subsequent discovery of a yield-boosting "missing link" carboxylesterase that selectively deprotects a late-stage intermediate, so triggering more efficient furan biosynthesis. This has allowed, for the first time, the isolation and structural elucidation of unknown intermediates, refining our understanding of furan formation in limonoid biosynthesis.

Spinning sugars in antigen biosynthesis: characterization of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases.

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.

Heterologous Expression of the Formicamycin Biosynthetic Gene Cluster Unveils Glycosylated Fasamycin Congeners.

Formicamycins and their biosynthetic intermediates the fasamycins are polyketide antibiotics produced by Streptomyces formicae KY5 from a pathway encoded by the for biosynthetic gene cluster. In this work the ability of Streptomyces coelicolor M1146 and the ability of Saccharopolyspora erythraea Δery to heterologously express the for biosynthetic gene cluster were assessed. This led to the identification of eight new glycosylated fasamycins modified at different phenolic groups with either a monosaccharide (glucose, galactose, or glucuronic acid) or a disaccharide comprised of a proximal hexose (either glucose or galactose), with a terminal pentose (arabinose) moiety. In contrast to the respective aglycones, minimal inhibitory screening assays showed these glycosylated congeners lacked antibacterial activity.

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates.

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.

Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity.

Glycosylation of small molecules is critical for numerous biological processes in plants, including hormone homeostasis, neutralization of xenobiotics, and synthesis and storage of specialized metabolites. Glycosylation of plant natural products is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits such as disease resistance and flavor and have numerous pharmaceutical applications. Most characterized plant natural product UGTs are glucosyltransferases, and little is known about enzymes that add other sugars. Here we report the discovery and characterization of AsAAT1 (UGT99D1), which is required for biosynthesis of the antifungal saponin avenacin A-1 in oat (Avena strigosa). This enzyme adds l-Ara to the triterpene scaffold at the C-3 position, a modification critical for disease resistance. The only previously reported plant natural product arabinosyltransferase is a flavonoid arabinosyltransferase from Arabidopsis (Arabidopsis thaliana). We show that AsAAT1 has high specificity for UDP-ß-l-arabinopyranose, identify two amino acids required for sugar donor specificity, and through targeted mutagenesis convert AsAAT1 into a glucosyltransferase. We further identify a second arabinosyltransferase potentially implicated in the biosynthesis of saponins that determine bitterness in soybean (Glycine max). Our investigations suggest independent evolution of UDP-Ara sugar donor specificity in arabinosyltransferases in monocots and eudicots.

Assessing the Toxicity and Mitigating the Impact of Harmful Prymnesium Blooms in Eutrophic Waters of the Norfolk Broads.

Prymnesium parvum is a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvum blooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies.

Discovery of an RmlC/D fusion protein in the microalga Prymnesium parvum and its implications for NDP-ß-l-rhamnose biosynthesis in microalgae.

The 6-deoxy sugar l-rhamnose (l-Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host-pathogen interactions often means that l-Rha plays an essential role in many organisms. l-Rha is most commonly biosynthesized as the activated sugar nucleotide uridine 5'-diphospho-ß-l-rhamnose (UDP-ß-l-Rha) or thymidine 5'-diphospho-ß-l-rhamnose (TDP-ß-l-Rha). Enzymes involved in the biosynthesis of these sugar nucleotides have been studied in some detail in bacteria and plants, but the activated form of l-Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using sugar-nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum, we show that levels of UDP- and TDP-activated l-Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-ß-l-Rha biosynthesis, from P. parvum Using this new sequence and also others, we explored l-Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like l-Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate ß-l-Rha (NDP-ß-l-Rha) pathways among algae.

Identification of a Kdn biosynthesis pathway in the haptophyte Prymnesium parvum suggests widespread sialic acid biosynthesis among microalgae.

Sialic acids are a family of more than 50 structurally distinct acidic sugars on the surface of all vertebrate cells where they terminate glycan chains and are exposed to many interactions with the surrounding environment. In particular, sialic acids play important roles in cell-cell and host-pathogen interactions. The sialic acids or related nonulosonic acids have been observed in Deuterostome lineages, Eubacteria, and Archaea but are notably absent from plants. However, the structurally related C8 acidic sugar 3-deoxy-d-manno-2-octulosonic acid (Kdo) is present in Gram-negative bacteria and plants as a component of bacterial lipopolysaccharide and pectic rhamnogalacturonan II in the plant cell wall. Until recently, sialic acids were not thought to occur in algae, but as in plants, Kdo has been observed in algae. Here, we report the de novo biosynthesis of the deaminated sialic acid, 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), in the toxin-producing microalga Prymnesium parvum Using biochemical methods, we show that this alga contains CMP-Kdn and identified and recombinantly expressed the P. parvum genes encoding Kdn-9-P synthetase and CMP-Kdn synthetase enzymes that convert mannose-6-P to CMP-Kdn. Bioinformatics analysis revealed sequences related to those of the two P. parvum enzymes, suggesting that sialic acid biosynthesis is likely more widespread among microalgae than previously thought and that this acidic sugar may play a role in host-pathogen interactions involving microalgae. Our findings provide evidence that P. parvum has the biosynthetic machinery for de novo production of the deaminated sialic acid Kdn and that sialic acid biosynthesis may be common among microalgae.

Identification of Euglena gracilis ß-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149.

Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. ß-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a ß-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a ß-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial ß-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.

Serine-rich repeat protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles.

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcß(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low ß-L-ODAP grass pea lines.

BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.

The Mechanism of Acetyl Transfer Catalyzed by Mycobacterium tuberculosis GlmU.

The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP- N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5'-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.

Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Šresolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.

Insights into toxic Prymnesium parvum blooms: the role of sugars and algal viruses.

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

A call to incorporate systems theory and human factors into the existing investigation of harm in clinical research involving healthcare products.

This is a joint statement from individual pharmacology and pharmaceutical professionals acting in their own capacity, including members of the Alliance for Clinical Research Excellence and Safety (ACRES) and the International Society of Pharmacovigilance (ISoP). By building on the extensive pharmacological and regulatory investigations that already take place, we are calling for a fuller and more robust systems-based approach to the independent investigation of clinical research when serious incidents of harm occur, starting with first-in-human clinical trials. To complement existing activities and regulations, we propose an additional approach blending evidence derived from both pharmacological and organizational science, which addresses human factors and transparency, to enhance organizational learning and continuous improvement. As happens with investigations in other sectors of society, such as the chemical and aviation sector, this systems approach should be seen as an additional way to understand how problems occur and how they might be prevented in the future. We believe that repetition of potentially preventable and adverse outcomes during clinical research, by failing to identify and act upon all systematic vulnerabilities, is a situation that needs urgent change. As we will discuss further on, approaches based on applying systems theory and human factors are much more likely to improve objectivity and transparency, leading to better system design.

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1).

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.

Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism.

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.