Glucan Branching Enzyme Research Articles

Characterization of Starch Physicochemical Properties and Grain Transcriptome Reveal the Mechanism for Resistant Starch Accumulation

Understanding the physicochemical properties of starch during grain development and the mechanism for resistant starch (RS) accumulation will provide useful information for improving the RS content of wheat. The grains from wheat mutant lines with high RS contents and their corresponding wild-type control were analyzed to characterize the structural and physicochemical properties of wheat starch. A transcriptomic analysis was used to analyze the differentially expressed genes (DEGs) involved in RS accumulation. The results showed that the RS content increased with grain development, along with the total starch content, but a larger increase was observed in the middle and later stages of grain filling. The X-ray diffraction peak intensity and relative crystallinity of starch exhibited the lowest and highest values at 10 days after anthesis, respectively. Regarding the thermal properties of starch, the peak temperature and conclusion temperature generally decreased with grain development; however, the enthalpy values showed no apparent regularity. Compared to control cultivar ZM22, the RS639 and RS683 lines with high RS contents showed high amylose contents and high relative crystallinity and a large proportion of 2.0~9.8 µm starch granules. Furthermore, the transcriptomics analysis revealed that the average relative expression of the glucan-branching enzyme (GBE) α-1,4 glucan phosphorylase (Pho) and starch synthase (SS) in ZM22 was 2.47-, 2.70-, and 2.56-fold higher than that in RS639, respectively; which indicates that the downregulation of the expression of genes encoding GBE, Pho, and SS in wheat grain promotes the accumulation of RS.

Characterization of the Glucan-Branching Enzyme GlgB Gene from Swine Intestinal Bacteria.

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.

Conserved residues Glu and Phe at substrate binding groove of α-1,6-glucanases modulate branch of the product

Understanding what amino acids in α-1,6-glucanases target α-1,6 glycosidic bonds of polysaccharides is timely and important for generating products with branch structure. With this objective, we investigated 330 sequences from seven subfamilies to excavate amino acids for recognition or catalysis of α-1,6 glycosidic bonds. Computational analysis identified two amino acids, E343 and W521, trigger α-1,6 glycosidic bond specificity of enzymes. To explore the effect of E343 and W521 on the product structure, several engineered mutants were studied in our research. Product structural analysis showed that the ratio of amylose and amylopectin is obviously different. The catalytic mechanism revealed that the bulky aromatic side chain is a trigger that controls the ratio of branch glucans. The E148 acts as a proton donor to regulate the generation of branched structures in the product during transglycosidation of the glucan branching enzyme (GBE).

Glycosyltransferases improve breadmaking quality by altering multiscale structure in gluten-free bread

In gluten-free bread (GFB), starch and its interplay with structural agents were vital for product quality due to the absence of gluten. During breadmaking, amylolytic enzymes modified starch in situ , and their reaction modes determined starch structure and hence how interactions occurred. Herein, based on the matrices of rice protein and hydroxypropyl methylcellulose (HPMC), α-amylase and multifunctional enzymes of glucan branching enzyme (GBE) and cyclodextrin glycosyltransferases (CGTase) were adopted, aiming to understand the mechanism of these enzymes on breadmaking quality of fresh bread from the multiscale structure of GFB. Results demonstrated that all enzymes, especially CGTase and GBE, markedly increased GFB specific volume to 107%–134% of Control. Compared with α-amylase, CGTase and GBE more depended on the structural changes rather than fermentation to improve GFB volume. Considering the texture, CGTase and GBE reduced crumb stickiness and still maintained a relatively softness and springiness crumb, while α-amylase induced a stick and less elastic crumb. Starch molecular changes enhanced the entanglement of starch and HPMC to form extended and bold fibril bundles and finally improved strength and pore size of gel network structure. Moreover, the starch structure modifications and specific enzymatic products of CGTase and GBE altered the microstructure and thus facilitated the even distribution of rice protein, which further improved GFB texture properties. Amylolytic enzymes, that acted both hydrolysis and glycosyl transfer, moderately hydrolyzed starch and generated specific enzymatic products, were more suitable to enhance GFB structure and eliminate the weak crumb texture. • α-amylase (α-Amy) and glycosyltransferases (GTases) were included. • Breadmaking quality of gluten-free bread (GFB) with α-Amy and GTases was compared. • GFB with GTases performed better in crumb structure and texture. • GTases depended more on structure rather than gas to improve volume. • Interactions of enzymatic starch and structural agents was crucial for GFB quality.

Highly branched starch accelerates the restoration of edible quality of dried rice noodles during rehydration

Rice noodle with excellent edible quality usually needs a dense gel network structure, but the dense structure is detrimental to the entry of water molecules during dried rice noodle rehydration. To combine the conflict requirements, we described the branching modification of rice starch using 1,4- α- glucan branching enzyme (GBE). Highly branched starch with a short cluster structure reduced fractal dimension and crystallite thickness while increasing mesh size and hydrophilicity of rice noodle gel network. Rice noodles derived from rice soaked with 128 U/g of GBE had a desirable rehydration time (370 s), which was reduced by 39.84% compared to the control. Meanwhile, the shorter double helix formed by the short cluster contributed to the improved short-range order, resulting in GBE-modified rice noodles with significantly higher tensile strength than control. These findings demonstrate that manipulating the branching degree of starch is an effective method for producing high-quality instant rice noodles.

Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13–24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G’) and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Introduction of glucan synthase into the cytosol in wheat endosperm causes massive maltose accumulation and represses starch synthesis.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic β-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of β-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

Transcriptomic, proteomic, and physiological comparative analyses of flooding mitigation of the damage induced by low-temperature stress in direct seeded early indica rice at the seedling stage

BackgroundLow temperature (LT) often occurs at the seedling stage in the early rice-growing season, especially for direct seeded early-season indica rice, and using flooding irrigation can mitigate LT damage in rice seedlings. The molecular mechanism by which flooding mitigates the damage induced by LT stress has not been fully elucidated. Thus, LT stress at 8 °C, LT accompanied by flooding (LTF) and CK (control) treatments were established for 3 days to determine the transcriptomic, proteomic and physiological response in direct seeded rice seedlings at the seedling stage.ResultsLT damaged chloroplasts, and thylakoid lamellae, and increased osmiophilic bodies and starch grains compared to CK, but LTF alleviated the damage to chloroplast structure caused by LT. The physiological characteristics of treated plants showed that compared with LT, LTF significantly increased the contents of rubisco, chlorophyll, PEPCK, ATP and GA3 but significantly decreased soluble protein, MDA and ABA contents. 4D-label-free quantitative proteomic profiling showed that photosynthesis-responsive proteins, such as phytochrome, as well as chlorophyll and the tricarboxylic acid cycle were significantly downregulated in LT/CK and LTF/CK comparison groups. However, compared with LT, phytochrome, chlorophyllide oxygenase activity and the glucan branching enzyme in LTF were significantly upregulated in rice leaves. Transcriptomic and proteomic studies identified 72,818 transcripts and 5639 proteins, and 4983 genes that were identified at both the transcriptome and proteome levels. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were significantly enriched in glycine, serine and threonine metabolism, biosynthesis of secondary metabolites, glycolysis/gluconeogenesis and metabolic pathways.ConclusionThrough transcriptomic, proteomic and physiological analyses, we determined that a variety of metabolic pathway changes were induced by LT and LTF. GO and KEGG enrichment analyses demonstrated that DEGs and DEPs were associated with photosynthesis pathways, antioxidant enzymes and energy metabolism pathway-related proteins. Our study provided new insights for efforts to reduce the damage to direct seeded rice caused by low-temperature stress and provided a breeding target for low temperature flooding-resistant cultivars. Further analysis of translational regulation and metabolites may help to elucidate the molecular mechanisms by which flooding mitigates low-temperature stress in direct seeded early indica rice at the seedling stage.

Enzymatic properties of an efficient glucan branching enzyme and its potential application in starch modification

Glucan branching enzymes (GBEs, EC 2.4.1.18) catalyze the formation of α-1,6-linked branch in starch, which is important for the starch modification with prospective properties. In this study, the aqGBE gene encoding an efficient glucan branching enzyme was cloned from Aquabacterium sp. strain A7-Y and successfully expressed in Escherichia coli BL21 (DE3). The specific activity of the purified recombinant enzyme rAqGBE was 2850 U/mg with potato starch as the optimal substrate, and the Km and Vmax values of rAqGBE were 1.18 mg/mL and 588.2 μmol/min/mg, respectively. Enzymological characterization showed that rAqGBE exhibits its optimal activity under the condition of 40 °C and pH 7.0, respectively, which is independent of calcium ions. Otherwise, rAqGBE-treated potato starch showed different chain length distribution compared with control, the numbers of short chains (degree of polymerization, DP < 7) and long chains (DP > 25) increased from 4.5% to 9.6% and 6.1%–15.7% after enzymatic treatment, respectively. In starch anti-ageing assay, with minimum usage of 0.8 mg rAqGBE per g starch, the rAqGBE-treated potato starch exhibited reduced retrogradation properties. Our results indicate that the branching enzyme AqGBE may therefore be a promising tool for the enzymatic modification of starch.

Effects of multienzyme treatment on the physicochemical properties of maize starch-lauric acid complex

A proposed strategy to increase the number of α-1,6 glycosidic bonds and the branch density of maize starch to some extent to promote the formation of a V-type complex by using an enzymatic method was studied. The molecular weight values were decreased after the starch underwent maltogenic amylase (MA)/glucan branching enzyme (GBE)/pullulanase (PU) treatment. The MA and MA→GBE modifications decrease the average chain length and increase the branch density of the starch. X-ray diffraction showed that the diffraction intensity of the inclusion complex became stronger after the starch was modified by dual- and triple-enzymes. The MA (30 min)-PU-starch-lauric acid (LA) complex had the highest melting enthalpy values among all of the analysed samples. FTIR indicated that there was more ordered crystalline structure formed in MA (30 min) and MA→GBE modified samples. The swelling power values of enzyme-treated starch-LA complex occurred in the following order: MA (60 min)/PU > MA/GBE/PU > PU > MA (30 min)/PU. In vitro digestibility results indicated that the enzyme resistance of the starch-LA complex was enhanced by applying the MA→PU and MA→GBE→PU modification to the starch.

Partial isolation of the starch branching enzymes [SBE] encoding gene from the plantain banana [Musa paradisiaca] genotype Raja

Banana fruit has the potential to be developed as a functional food in terms of its medicinal benefit. One of its features enabling such beneficial uses is its resistant starch characteristic. However, the starch resistance composition of the fruit will decrease during fruit maturation and food processing. Rationally, blocking of starch resistance decreasing process should maintain the beneficial characteristic of the banana fruit. This could be done by suppressing the expression of the SBE gene so that it can inhibit the formation of branching points in the amylopectin chain metabolism. Based on that consideration, we isolated the SBE gene involved in the formation of amylopectin in genotype Pisang Raja. In the initial step, the MaSBE_5458_F and MaSBE_5458_R primer combination was designed and successfully generated part of the sequence of the MaSBE5 gene. A total DNA sequence of 1,372 bp in length has been isolated, purified and characterized by sequencing technique. Blast analysis showed that the MaSBE_5458 gene sequence has a 97% similarity with 1,4-alpha glucan branching enzyme 1, chloroplastic/amyloplastic DNA sequence isolated from Musa acuminata subsp. malaccensis.

Prolyl hydroxylase inhibition protects the kidneys from ischemia via upregulation of glycogen storage

Hypoxia-inducible factor (HIF) mediates protection via hypoxic preconditioning in both, invitro and invivo ischemia models. However, the underlying mechanism remains largely unknown. Prolyl hydroxylase domain proteins serve as the main HIF regulator via hydroxylation of HIFα leading to its degradation. At present, prolyl hydroxylase inhibitors including enarodustat are under clinical trials for the treatment of renal anemia. In an invitro model of ischemia produced by oxygen-glucose deprivation of renal proximal tubule cells in culture, enarodustat treatment and siRNA knockdown of prolyl hydroxylase 2, but not of prolyl hydroxylase 1 or prolyl hydroxylase 3, significantly increased the cell viability and reduced the levels of reactive oxygen species. These effects were offset by the simultaneous knockdown of HIF1α. In another invitro ischemia model induced by the blockade of oxidative phosphorylation with rotenone/antimycin A, enarodustat-enhanced glycogen storage prolonged glycolysis and delayed ATP depletion. Although autophagy is another possible mechanism of prolyl hydroxylase inhibition-induced cytoprotection, gene knockout of a key autophagy associated protein, Atg5, did not affect the protection. Enarodustat increased the expression of several enzymes involved in glycogen synthesis, including phosphoglucomutase 1, glycogen synthase 1, and 1,4-α glucan branching enzyme. Increased glycogen served as substrate for ATP and NADP production and augmented reduction of glutathione. Inhibition of glycogen synthase 1 and glutathione reductase nullified enarodustat's protective effect. Enarodustat also protected the kidneys in a rat ischemia reperfusion injury model and the protection was partially abrogated by inhibiting glycogenolysis. Thus, prolyl hydroxylase inhibition protects the kidney from ischemia via upregulation of glycogen synthesis.

Metagenomic reconstructions of gut microbial metabolism in weanling pigs

BackgroundThe piglets’ transition from milk to solid feed induces a succession of bacterial communities, enhancing the hosts’ ability to harvest energy from dietary carbohydrates. To reconstruct microbial carbohydrate metabolism in weanling pigs, this study combined 16S rRNA gene sequencing (n = 191) and shotgun metagenomics (n = 72).ResultsTime and wheat content in feed explained most of the variation of the microbiota as assessed by 16S rRNA gene sequencing in weanling pigs. De novo metagenomic binning reconstructed 360 high-quality genomes that represented 11 prokaryotic and 1 archaeal phylum. Analysis of carbohydrate metabolism in these genomes revealed that starch fermentation is carried out by a consortium of Firmicutes expressing extracellular α-(1 → 4)-glucan branching enzyme (GH13) and Bacteroidetes expressing periplasmic neopullulanase (GH13) and α-glucosidase (GH97). Fructans were degraded by extracellular GH32 enzymes from Bacteriodetes and Lactobacillus. Lactose fermentation by β-galactosidases (GH2 and GH42) was identified in Firmicutes. In conclusion, the assembly of 360 high-quality genomes as the first metagenomic reference for swine intestinal microbiota allowed identification of key microbial contributors to degradation of starch, fructans, and lactose.ConclusionsMicrobial consortia that are responsible for degradation of these glycans differ substantially from the microbial consortia that degrade the same glycans in humans. Our study thus enables improvement of feeding models with higher feed efficiency and better pathogen control for weanling pigs.

Distribution of glucan-branching enzymes among prokaryotes.

Glucan-branching enzyme plays an essential role in the formation of branched polysaccharides, glycogen, and amylopectin. Only one type of branching enzyme, belonging to glycoside hydrolase family 13 (GH13), is found in eukaryotes, while two types of branching enzymes (GH13 and GH57) occur in prokaryotes (Bacteria and Archaea). Both of these types are the members of protein families containing the diverse specificities of amylolytic glycoside hydrolases. Although similarities are found in the catalytic mechanism between the two types of branching enzyme, they are highly distinct from each other in terms of amino acid sequence and tertiary structure. Branching enzymes are found in 29 out of 30 bacterial phyla and 1 out of 5 archaeal phyla, often along with glycogen synthase, suggesting the existence of α-glucan production and storage in a wide range of prokaryotes. Enormous variability is observed as to which type and how many copies of branching enzyme are present depending on the phylum and, in some cases, even among species of the same genus. Such a variation may have occurred through lateral transfer, duplication, and/or differential loss of genes coding for branching enzyme during the evolution of prokaryotes.

Molecular Genetic Analysis of Glucan Branching Enzymes from Plants and Bacteria in Arabidopsis Reveals Marked Differences in Their Functions and Capacity to Mediate Starch Granule Formation.

The major component of starch is the branched glucan amylopectin, the branching pattern of which is one of the key factors determining its ability to form semicrystalline starch granules. Here, we investigated the functions of different branching enzyme (BE) types by expressing proteins from maize (Zea mays BE2a), potato (Solanum tuberosum BE1), and Escherichia coli (glycogen BE [EcGLGB]) in Arabidopsis (Arabidopsis thaliana) mutant plants that are deficient in their endogenous BEs and therefore, cannot make starch. The expression of each of these three BE types restored starch biosynthesis to differing degrees. Full complementation was achieved using the class II BE ZmBE2a, which is most similar to the two endogenous Arabidopsis isoforms. Expression of the class I BE from potato, StBE1, resulted in partial complementation and high amylose starch. Expression of the glycogen BE EcGLGB restored only minimal amounts of starch production, which had unusual chain length distribution, branch point distribution, and granule morphology. Nevertheless, each type of BE together with the starch synthases and debranching enyzmes were able to create crystallization-competent amylopectin polymers. These data add to the knowledge of how the properties of the BE influence the final composition of starch and fine structure of amylopectin.

Diversity of reaction characteristics of glucan branching enzymes and the fine structure of α-glucan from various sources

To investigate the functional properties of 10 α-glucan branching enzymes (BEs) from various sources, we determined the chain-length distribution of BE enzymatic products and their phosphorylase-limit dextrins (Φ-LD). All BEs could be classified into either of the three rice BE isozymes: OsBEI, OsBEIIa, or OsBEIIb. Escherichia coli BE (EcoBE) had the same enzymatic properties as OsBEI, while Synechococcus elongatus BE (ScoBE) and Chlorella kessleri BE (ChlBE) had BEIIb-type properties. Human BE (HosBE), yeast BE (SacBE), and two Porphyridium purpureum BEs (PopBE1 and PopBE2) exhibited the OsBEIIa-type properties. Analysis of chain-length profile of Φ-LD of the BE reaction products revealed that EcoBE, ScoBE, PopBE1, and PopBE2 preferred A-chains as acceptors, while OsBEIIb used B-chains more frequently than A-chains. Both EcoBE and ScoBE specifically formed the branch linkages at the third glucose residue from the reducing end of the acceptor chain. The present results provide evidence for the first time that great variation exists as to the preference of BEs for their acceptor chain, either A-chain or B-chain. In addition, EcoBE and ScoBE recognize the location of branching points in their acceptor chain during their branching reaction. Nevertheless, no correlation exists between the primary structure of BE proteins and their enzymatic characteristics.

Gene-targeted metagenomic analysis of glucan-branching enzyme gene profiles among human and animal fecal microbiota.

Glycoside hydrolases (GHs), the enzymes that breakdown complex carbohydrates, are a highly diversified class of key enzymes associated with the gut microbiota and its metabolic functions. To learn more about the diversity of GHs and their potential role in a variety of gut microbiomes, we used a combination of 16S, metagenomic and targeted amplicon sequencing data to study one of these enzyme families in detail. Specifically, we employed a functional gene-targeted metagenomic approach to the 1-4-α-glucan-branching enzyme (gBE) gene in the gut microbiomes of four host species (human, chicken, cow and pig). The characteristics of operational taxonomic units (OTUs) and operational glucan-branching units (OGBUs) were distinctive in each of hosts. Human and pig were most similar in OTUs profiles while maintaining distinct OGBU profiles. Interestingly, the phylogenetic profiles identified from 16S and gBE gene sequences differed, suggesting the presence of different gBE genes in the same OTU across different vertebrate hosts. Our data suggest that gene-targeted metagenomic analysis is useful for an in-depth understanding of the diversity of a particular gene of interest. Specific carbohydrate metabolic genes appear to be carried by distinct OTUs in different individual hosts and among different vertebrate species' microbiomes, the characteristics of which differ according to host genetic background and/or diet.

Hypoxia Promotes Glycogen Accumulation through Hypoxia Inducible Factor (HIF)-Mediated Induction of Glycogen Synthase 1

When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide

BackgroundMycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1.ResultsUsing yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C40S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific.ConclusionWe conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1.

The evolution of putative starch-binding domains

The present bioinformatics analysis was focused on the starch-binding domains (SBDs) and SBD-like motifs sequentially related to carbohydrate-binding module (CBM) families CBM20 and CBM21. Originally, these SBDs were known from microbial amylases only. At present homologous starch- and glycogen-binding domains (or putative SBD sequences) have been recognised in various plant and animal proteins. The sequence comparison clearly showed that the SBD-like sequences in genethonin-1, starch synthase III and glucan branching enzyme should possess the real SBD function since the two tryptophans (or at least two aromatics) of the typical starch-binding site 1 are conserved in their sequences. The same should apply also for the sequences corresponding with the so-called KIS-domain of plant AKINβγ protein that is a homologue of the animal AMP-activated protein kinase (AMPK). The evolutionary tree classified the compared SBDs into three distinct groups: (i) the family CBM20 (the motifs from genethonins, laforins, starch excess 4 protein, β-subunits of the animal AMPK and all plant and yeast homologues, and eventually from amylopullulanases); (ii) the family CBM21 (the motifs from regulatory subunits of protein phosphatase 1 together with those from starch synthase III); and (iii) the (CBM20 + CBM21)-related group (the motifs from the pullulanase subfamily consisting of pullulanase, branching enzyme, isoamylase and maltooligosyl trehalohydrolase).