A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping

Uracil DNA glycosylase (UNG) is the primary enzyme for the removal of uracil from the genome of many organisms. A key question is how the enzyme is able to scan large quantities of DNA in search of aberrant uracil residues. Central to this is the mechanism by which it flips the target nucleotide out of the DNA helix and into the enzyme-active site. Both active and passive mechanisms have been proposed. Here, we report a rapid kinetic analysis using two fluorescent chromophores to temporally resolve DNA binding and base-flipping with DNA substrates of different sequences. This study demonstrates the importance of the protein–DNA interface in the search process and indicates an active mechanism by which UNG glycosylase searches for uracil residues

A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (kcat/Km) compared with the viral enzyme due to a lower Km. The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2′-deoxypseudouridine and 2′-α-fluoro-2′-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U·A and U·G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair

Recognition and repair of DNA damage by uracil DNA glycosylase

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Yield, Grain Quality, and Starch Physicochemical Properties of 2 Elite Thai Rice Cultivars Grown under Varying Production Systems and Soil Characteristics

Rice production systems and soil characteristics play a crucial role in determining its yield and grain quality. Two elite Thai rice cultivars, namely, KDML105 and RD6, were cultivated in two production systems with distinct soil characteristics, including net-house pot production and open-field production. Under open-field system, KDML105 and RD6 had greater panicle number, total grain weight, 100-grain weight, grain size, and dimension than those grown in the net-house. The amounts of reducing sugar and long amylopectin branch chains (DP 25–36) of the RD6 grains along with the amounts of long branch chains (DP 25–36 and DP ≥ 37), C-type starch granules, and average chain length of the KDML105 were substantially enhanced by the open-field cultivation. Contrastingly, the relative crystallinity of RD6 starch and the amounts of short branch chains (DP 6–12 and DP 13–24), B- and A-type granules, and median granule size of KDML105 starch were significantly suppressed. Consequently, the open-field-grown RD6 starch displayed significant changes in its gelatinization and retrogradation properties, whereas, certain retrogradation parameters and peak viscosity (PV) of KDML105 starches were differentially affected by the distinct cultivating conditions. This study demonstrated the influences of production systems and soil characteristics on the physicochemical properties of rice starches

Production of Large-Ring Cyclodextrins by Amylomaltases

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60–70 °C for thermostable amylomaltases and 30–45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0–7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases

Computational design of Lactobacillus Acidophilus α-L-rhamnosidase to increase its structural stability.

α-L-rhamnosidase catalyzes hydrolysis of the terminal α-L-rhamnose from various natural rhamnoglycosides, including naringin and hesperidin, and has various applications such as debittering of citrus juices in the food industry and flavonoid derhamnosylation in the pharmaceutical industry. However, its activity is lost at high temperatures, limiting its usage. To improve Lactobacillus acidophilus α-L-rhamnosidase stability, we employed molecular dynamics (MD) to identify a highly flexible region, as evaluated by its root mean square fluctuation (RMSF) value, and computational protein design (Rosetta) to increase rigidity and favorable interactions of residues in highly flexible regions. MD results show that five regions have the highest flexibilities and were selected for design by Rosetta. Twenty-one designed mutants with the best ΔΔG at each position and ΔΔG < 0 REU were simulated at high temperature. Eight designed mutants with ΔRMSF of highly flexible regions lower than -10.0% were further simulated at the optimum temperature of the wild type. N88Q, N202V, G207D, Q209M, N211T and Y213K mutants were predicted to be more stable and could maintain their native structures better than the wild type due to increased hydrogen bond interactions of designed residues and their neighboring residues. These designed mutants are promising enzymes with high potential for stability improvement

Substrates 1HU (left column) and 2HU (right column) were mixed with increasing concentrations of D88N/H210N UNG using stopped-flow

<p><b>Copyright information:</b></p><p>Taken from "A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping"</p><p></p><p>Nucleic Acids Research 2007;35(5):1478-1487.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865060.</p><p>© 2007 The Author(s).</p> Anisotropy ( and ) and total HEX fluorescence ( and ) were simultaneously monitored, and the same solutions were then used to collect 2-AP fluorescence ( and ). The data are shown with the results of a global fit to Scheme 1. Individual curves for each of the enzyme concentrations used are shown: 8 μM (red); 3 μM (green); 2 μM (blue); 1 μM (cyan); 0.5 μM (magenta) and 0.2 μM (purple), all reactions were performed with 0.1 μM substrate and other conditions as described in the Materials and methods section

A complete reaction cycle of UNG was analysed by monitoring 2-AP fluorescence using stopped-flow to rapidly mixing equimolar amounts of wtUNG and substrates 1U () and 2U () at concentrations in excess of the (4 μM 1U and 20 μM 2U)

<p><b>Copyright information:</b></p><p>Taken from "A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping"</p><p></p><p>Nucleic Acids Research 2007;35(5):1478-1487.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865060.</p><p>© 2007 The Author(s).</p> The data are shown with the best fit to Scheme 1 using kinetic parameters determined from the global stopped-flow analysis (), the cleavage rate determined from the quench-flow analysis (), and fitting only a single kinetic parameter, the off-rate (), together with the fluorescence coefficients for substrate (), enzyme–substrate complex () and product (; )

Insight into the Molecular Weight of Hydrophobic Starch Laurate-Based Adhesives for Paper

Instead of using finite petroleum-based resources and harmful additives, starch can be used as a biodegradable, low-cost, and non-toxic ingredient for green adhesives. This work employs K3PO4 catalyzed transesterifications of cassava starch and methyl laurate at varying reaction times (1–10 h), resulting in the enhanced hydrophobicity of starch laurates. At longer reaction times, starch laurates having higher degrees of substitution (DS) were obtained. While starch laurates are the major products of transesterification, relatively low-molecular-weight byproducts (1%) were detected and could be hydrolyzed starches based on gel permeation chromatography results. Contact angle measurements confirmed the relatively high hydrophobicity of the modified starches compared with that of native starch. The modified starches were then employed to prepare water-based adhesives on paper (without any additional additives). Notably, the shear strength of the esterified starch adhesives appears to be independent of the DS of esterified samples, hence the transesterification reaction times. Additionally, the shear strength of water-based adhesives (0.67–0.73 MPa) for bonding to paper substrates is superior to that of two other commercially available glues by a factor of 10 to 80 percent

Identification of crucial amino acid residues involved in large ring cyclodextrin synthesis by amylomaltase from Corynebacterium glutamicum

Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of Corynebacterium glutamicum amylomaltase (CgAM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated CgAM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of CgAM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the kcat of disproportionation activity, while E231Y mutant exhibited much higher kcat and Km values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations’ impacts on the CgAM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs