β-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.     

Phylogenetic analysis and inflow route of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> (TYLCV) and <Emphasis Type="Italic">Bemisia tabaci</Emphasis> in Korea

Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus of the family Geminiviridae, members of which are characterized by closed circular single-stranded DNA genomes of 2.7-2.8 kb in length, and include viruses transmitted by the Bemisia tabaci whitefly. No reports of TYLCV in Korea are available prior to 2008, after which TYLCV spread rapidly to most regions of the southern Korean peninsula (Gyeongsang-Do, Jeolla-Do and Jeju-Do). Fifty full sequences of TYLCV were analyzed in this study, and the AC1, AV1, IR, and full sequences were analyzed via the muscle program and bayesian analysis. Phylogenetic analysis demonstrated that the Korea TYLCVs were divided into two subgroups. The TYLCV Korea 1 group (Masan) originated from TYLCV Japan (Miyazaki) and the TYLCV Korea 2 group (Jeju/Jeonju) from TYLCV Japan (Tosa/Haruno). A B. tabaci phylogenetic tree was constructed with 16S rRNA and mitochondria cytochrome oxidase I (MtCOI) sequences using the muscle program and MEGA 4.0 in the neighbor-joining algorithm. The sequence data of 16S rRNA revealed that Korea B. tabaci was closely aligned to B. tabaci isolated in Iran and Nigeria. The Q type of B. tabaci, which was originally identified as a viruliferous insect in 2008, was initially isolated in Korea as a non-viruliferous insect in 2005. Therefore, we suggest that two TYLCV Japan isolates were introduced to Korea via different routes, and then transmitted by native B. tabaci.     

Enzymatic preparation of maltohexaose, maltoheptaose, and maltooctaose by the preferential cyclomaltooligosaccharide (cyclodextrin) ring-opening reaction of Pyrococcus furiosus thermostable amylase

Specific-length maltooligosaccharides, particularly maltohexaose, maltoheptaose, and maltooctaose, were prepared from cyclomaltooligosaccharides (cyclodextrins, CDs) by the preferential cyclodextrin ring-opening reaction of an amylolytic enzyme from Pyrococcus furiosus. The enzyme primarily produces maltohexaose, maltoheptaose, and maltooctaose by hydrolyzing alpha-, beta-, and gamma-CD, respectively. This study aims to develop a high-efficiency synthesis of specific maltooligosaccharides at high-purity. [formula: see text]     

Enzymatic synthesis of dimaltosyl-beta-cyclodextrin via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme

Di-O-α-maltosyl-β-cyclodextrin ((G2)₂-β-CD) was synthesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-β-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-β-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-β-CD, suggesting that TreX transferred the maltosyl residue of a G2-β-CD to another molecule of G2-β-CD by forming an α-1,6-glucosidic linkage. When [¹⁴C]-maltose and maltosyl-β-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-β-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6¹,6³- and 6¹,6⁴-dimaltosyl-β-CD. Inhibition studies with β-CD on the transglycosylation activity revealed that β-CD was a mixed-type inhibitor, with a K_i value of 55.6 μmol/mL. Thus, dimaltosyl-β-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of β-CD. Our findings suggest that the high yield of (G2)₂-β-CD from G2-β-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of β-CD.     

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and β-cyclodextrin (β-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and β-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched β-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the α-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.Bacillus subtilis can utilize polysaccharides such as starch, glycogen, and amylose as carbon sources by hydrolyzing them into smaller maltodextrins via the action of extracellular α-amylase (AmyE) (14). In B. subtilis, α-glucosidase encoded by malL has been known to contribute to maltodextrin metabolism in the cell (40, 41). Schönert et al. (42) reported that maltose is transported by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in B. subtilis. They also reported that maltodextrins with degrees of polymerization (DP) of 3 to 7 (G3 to G7) are taken up via a maltodextrin-specific (Mdx) ATP-binding cassette (ABC) transport system (42). This system is made up of a maltodextrin-binding protein (MdxE) and two membrane proteins (MdxF and MdxG), as well as an ATPase (MsmX). The basic model proposed for the transport and metabolism of maltooligosaccharides includes a series of carbohydrate-hydrolyzing and -transferring enzymes. However, the enzymatic hydrolysis of maltodextrins and glycogen, providing a major energy reservoir in prokaryotes, was not reflected in the model, due probably to a lack of experimental analysis. Unlike those in Bacillus spp., the transport and metabolic systems for maltodextrins in Escherichia coli have been investigated extensively (7, 9, 10). A model for maltose metabolism involving an α-glucanotransferase (MalQ), a maltodextrin glucosidase (MalZ), and a maltodextrin phosphorylase (MalP) was proposed previously based on analyses of the breakdown of ¹⁴C-labeled maltodextrins in various knockout mutants (10).Ninety bacterial genomes were analyzed to identify the enzymes involved in sugar metabolism, and the results suggested that bacterial enzymes for the synthesis and degradation of glycogen belong to the glucosyltransferase and glycosidase/transglycosidase families, respectively. Free-living bacteria such as B. subtilis carry a minimal set of enzymes for glycogen metabolism, encoded by the glg operon of five genes. The four genes most proximal to the promoter encode enzymes for the synthesis of glycogen, including a branching enzyme (glgB), an ADP-glucose phyrophosphorylase (glgC and glcD), and a glycogen synthase (glgA). On the other hand, the most distal gene, glgP, encodes a glycogen phosphorylase (a member of glycosyltransferase family 35) (13, 18), which degrades glycogen branches by forming glucose-1-phosphate (glucose-1-P). B. subtilis carries two additional enzymes encoded at separate loci, a maltogenic amylase (MAase [YvdF, encoded at 304°]) and a pullulanase (AmyX, encoded at 262°), which have been known to degrade glycogen in vitro (15, 31). These two enzymes are ubiquitous among Bacillus spp. and may play an important role in glycogen and maltodextrin metabolism in the bacteria (see Table S1 in the supplemental material).The MAase YvdF in B. subtilis 168 and its homologue in B. subtilis SUH4-2 share 99% identity at both the nucleotide and amino acid sequence levels (4). MAase (EC 3.2.1.133) is a multisubstrate enzyme that acts on substrates such as cyclodextrin (CD), maltooligosaccharides, pullulan, starch, and glycogen (4). MAase belongs to a subfamily of glycoside hydrolase family 13, along with cyclodextrinase (EC 3.2.1.54), neopullulanase (EC 3.2.1.135), and Thermoactinomyces vulgaris R-47 α-amylase II (46). Although the catalytic properties and tertiary structure of MAase have been studied extensively (33), its physiological role in the bacterial cell is yet to be elucidated. The expression pattern of MAase in B. subtilis 168 has been investigated by monitoring the β-galactosidase activity expressed from the yvdF promoter in defined media containing various carbon sources (20). The yvdF promoter is induced in medium containing maltose, starch, or β-CD but is repressed in the presence of glucose, fructose, sucrose, or glycerol as the sole carbon source. In a previous study, Spo0A, a master regulator determining the life cycle of B. subtilis, was shown to be related to the expression of MAase in a positive manner (20). Kiel et al. (18) reported that the glycogen operon in B. subtilis is turned on during sporulation by RNA polymerase containing σ^E. This finding indicated that MAase, along with glycogen phosphorylase and pullulanase, might be involved in the metabolism of maltodextrin and glycogen in vivo.Pullulanases are capable of hydrolyzing the α-1,6-glycosidic linkages of pullulan to form maltotriose (2, 11, 15, 28, 31, 38). In particular, type I pullulanases have been reported to hydrolyze the α-1,6-glycosidic linkages in branched oligosaccharides such as starch, amylopectin, and glycogen, forming maltodextrins linked by α-1,4-glycosidic linkages (11). Pullulanase is also known as a debranching enzyme. The enzymatic properties and three-dimensional structure of AmyX from B. subtilis 168 were investigated by Malle et al. (28). However, to date, the physiological function of pullulanase encoded by amyX has not been investigated.The aim of this study was to elucidate the physiological functions of MAase and pullulanase, specifically concentrating on their roles in the degradation of maltodextrin and glycogen in B. subtilis. For this purpose, studies of the localization of the enzymes, the accumulation of glycogen, and the distribution of glycogen side chains were performed using the wild type and knockouts of MAase- and pullulanase-related genes.     

Exogenous cytokinin treatment maintains cyclin homeostasis in rice seedlings that show changes of cyclin expression when the photoperiod is rapidly changed.

Cyclin is a fundamental regulator of the plant cell cycle. Five different types of cyclin genes (the A-, B-, C-, D-, and H-types) have been reported in Oryza sativa. However, except for Os;cycA1;1, Os;cycB2;1, and Os;cycB2;2, the mechanisms of expression of these cyclin genes have not yet been studied. The interactions of cyclins with cytokinin, an important trigger for cell cycle regulation, have also not been well studied. Here we used semi-quantitative RT-PCR in rice seedlings to analyze the effect of cytokinin on photomorphogenesis and the expression of six cyclin genes. Fifteen-day-old seedlings were grown in a 16/8 h light/dark cycle and then transferred to either constant light or constant dark. The expression of all the cyclin genes tested, except the C-type, decreased after 1 hour in the dark, but did not change after transfer to the light or when kinetin was added to the medium. Similarly, seedlings grown in the dark had decreased expression of the cyclin genes, except Os;cycB2;2, after transfer to the light, a decrease that was prevented by kinetin treatment. Thus, exogenous cytokinin plays an important role in maintaining homeostasis of cyclin gene expression following rapid changes of photoperiod.     

Hymenobacter swuensis sp. nov., a Gamma-Radiation-Resistant Bacteria Isolated from Mountain Soil

Gram stain-negative and non-motile bacteria, designated as DY53^T and DY43, were isolated from mountain soil in South Korea prior exposure with 5 kGy gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strains belonged to the family Cytophagaceae in the class Cytophagia. 16S rRNA gene sequence similarity of strains DY53^T and DY43 was 100 %. The highest degrees of sequence similarities of strains DY53^T and DY43 were found with Hymenobacter perfusus A1-12^T (98.8 %), Hymenobacter rigui WPCB131^T (98.5 %), H. yonginensis HMD1010^T (97.9 %), H. xinjiangensis X2-1g^T (96.6 %), and H. gelipurpurascens Txg1^T (96.5 %). The DNA G+C content of the novel strains DY53^T and DY43 were 59.5 mol%. Chemotaxonomic data revealed that strains possessed major fatty acids such as C_15:0 iso, C_15:0 anteiso, C_16:1 ω5c, summed feature 3 (16:1 ω7c/ω6c), summed feature 4 (17:1 anteiso B/iso I) and C_17:0 iso, and major polar lipid was phosphatidylethanolamine. The novel strains showed resistance to gamma radiation, with a D10 value (i.e., the dose required to reduce the bacterial population by tenfold) in excess of 5 kGy. Based on these data, strains DY53^T and DY43 should be classified as representing a novel species, for which the name Hymenobacter swuensis sp. nov. is proposed, with the type strain DY53^T (=KCTC 32018^T = JCM 18582^T) and DY43 (=KCTC 32010).