Unraveling the effect of ultrasound-assisted alkali treatment on β-lactoglobulin-flavonoids covalent complex system formation and its potential for lipophilic bioactive compounds delivery.

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone

Effect of phlorotannins modification on the physicochemical, structural and functional properties of soybean protein isolate and controlled hydrolysates: Covalent and non-covalent interactions

Modification of structural and functional characteristics of casein treated with quercetin via two interaction modes: Covalent and non-covalent interactions

Stability and bioavailability of β-carotene-loaded emulsions improved by covalent interactions between soybean protein isolate, myricetin, and β-glucan.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.

The interaction of tRNA (m5U54)-methyltransferase (RUMT) with in vitro synthesized unmodified tRNA and a 17-base oligoribonucleotide analog of the T-arm of tRNA in the absence of AdoMet has been investigated. Binary complexes are formed which are isolable on nitrocellulose filters and are composed of noncovalent and covalent complexes in nearly equal amounts. The covalent RUMT-RNA complexes are stable to SDS-PAGE and migrate slower than free enzyme or RNA. Kinetic and thermodynamic constants involved in formation and disruption of noncovalent and covalent binary complexes have been determined and interpreted in the context of steady-state kinetic parameters of the enzyme-catalyzed methylation and 5-H exchange of substrate. The results show that the isolable covalent complex is kinetically incompetent as an intermediate for methylation. Isotope trapping experiments show that when AdoMet is added to preformed binary complex, all bound tRNA is converted to methylated product; thus, the covalent complexes are chemically competent to form products. We have concluded that, after a reversible binary complex is formed, the catalytic thiol adds to the 6-carbon of the U54 of tRNA. The initial adduct leaves the reaction pathway to protonation at carbon 5; the latter can deprotonate and re-enter the pathway to form methylated product. It is speculated that covalent binary RUMT-RNA adducts may serve as depots of enzyme-tRNA complexes primed for methylation, or in unknown roles with RNAs other than tRNA.

The effect of derivatives of folic acid on the fluorodeoxyuridylate-thymidylate synthetase covalent complex in human colon xenografts

Revealing the non-enzymatic covalent interaction between neo-/crypto-chlorogenic acid and beta-lactoglobulin under nonthermal process and potential delivery capability.

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.

Effects of catechin types found in tea polyphenols on the structural and functional properties of soybean protein isolate–catechin covalent complexes

Lycopene is a promising biological functional component with various biological activities and excellent pharmacological activities. However, its low water solubility and stability lead to low oral bioavailability, which limits its edible and medicinal research. Then, it is necessary to explore effective methods to protect lycopene from destruction and further exploit its potential benefits. The absorption of lycopene in vivo is affected by solubility, stability, isomer type, emulsifying ability, difficulty in forming micelles in vivo, and interaction with food components. Emulsions, pickering emulsions, micelles, liposomes, bigels, beasds, solid dispersions, microcapsules, nanoparticles, electrospinning and other drug delivery systems can be used as good strategies to improve the stability and bioavailability of lycopene. In this paper, the absorption process of lycopene in vivo and the factors affecting its bioavailability were discussed, and the preparation strategies for improving the stability, bioavailability, and health benefits of lycopene were reviewed, to provide some clues and references for the full utilization of lycopene in the field of health. However, there are still various unresolved mysteries regarding the metabolism of lycopene. The safety and in vivo studies of various preparations should be further explored, and the above technologies also face the challenge of industrial production.

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.

Establishing a novel covalent complex of wheat gluten with tea polyphenols: Structure, digestion, and action mechanism

The modification of β-conglycinin by Polygonatum sibiricum polysaccharide through covalent and non-covalent interactions: structure and processing characteristics.

Currently, there is a need to explore the effects of different types of protein-anthocyanin complexations, as well as the possible changes in the nutrition and allergenicity of the formed complexes. Here, we systematically investigated the covalent and non-covalent interactions between cyanidin-3-O-glucoside (C3G) and two major milk proteins, α-casein (α-CN) and β-lactoglobulin (β-LG). Fluorescence quenching data showed that, under non-covalent conditions, C3G quenched the fluorescence of the two proteins via a static process, with the interaction forces being revealed; for covalent products, decreased fluorescence intensities were observed with red shifts in the λmax. Multiple spectroscopic analyses implied that C3G-addition induced protein structural unfolding through transitions between the random coil and ordered secondary components. With a two-stage simulated gastrointestinal (GI) digestion model, it was seen that covalent complexes, not their non-covalent counterparts, showed reduced protein digestibility, ascribed to structural changes resulting in the unavailability of enzyme cleaving sites. The GI digests displayed prominent 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging abilities (3.8-11.1 mM Trolox equivalents per mL digest), in contrast to the markedly reduced 1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacities. Additionally, covalent protein-C3G complexes, but not their non-covalent counterparts, showed lower IgE-binding levels in comparison to the native control. This study provides new understanding for the development of anthocyanin-milk protein systems as functional ingredients with health-beneficial properties.