Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state

Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase–nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5′-ended strand and its slower RecB helicase on the 3′-ended strand. At Chi hotspots (5′ GCTGGTGG 3′), RecB’s nuclease cuts the 3′-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB’s ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

Biomimetic CO Oxidation Below – 100 ⁰C by a Nitrate-Containing Metal-Free Microporous System

<p>CO oxidation is of importance both for organic and inorganic systems. Transition and precious metals on various supports can oxidize CO to CO₂. Among them, few systems, like Au/TiO₂, can perform CO oxidation at the low temperature of -70 ⁰C. Living (an)aerobic organisms perform CO oxidation with nitrate using complex enzymes under ambient temperatures which is an important pathway of their living cycle that enables them to “breathe”/produce energy in the absence of oxygen and leads to the carbonate mineral formation. Herein, we report that CO can be oxidized to CO₂ by nitrate at –140 ⁰C in completely inorganic system (zeolite) without metals. The transformation of NO_x and CO species in zeolite as well as the origin of this unique activity (catalyzed by Bronsted acid sites) are clarified using spectroscopic and computational approach.</p>

Biomimetic CO Oxidation Below – 100 ⁰C by a Nitrate-Containing Metal-Free Microporous System

<p>CO oxidation is of importance both for organic and inorganic systems. Transition and precious metals on various supports can oxidize CO to CO₂. Among them, few systems, like Au/TiO₂, can perform CO oxidation at the low temperature of -70 ⁰C. Living (an)aerobic organisms perform CO oxidation with nitrate using complex enzymes under ambient temperatures which is an important pathway of their living cycle that enables them to “breathe”/produce energy in the absence of oxygen and leads to the carbonate mineral formation. Herein, we report that CO can be oxidized to CO₂ by nitrate at –140 ⁰C in completely inorganic system (zeolite) without metals. The transformation of NO_x and CO species in zeolite as well as the origin of this unique activity (catalyzed by Bronsted acid sites) are clarified using spectroscopic and computational approach.</p>

Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

ABSTRACTCoordination of multiple activities of complex enzymes is critical for life, including transcribing, replicating, and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5’-ended strand and its slower RecB helicase on the 3’-ended strand. At Chi hotspots (5’GCTGGTGG3’), RecB’s nuclease cuts the 3’-ended strand and loads RecA strand-exchange protein onto it. We report here that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate’s length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA distal end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. Computation docks NSAC1003 into the ATP-binding site, suggesting that NSAC1003 acts directly on RecB. NSAC1003 will help elucidate the molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes whose activities are coordinated at chromosomal sites.

Effects of Complex Enzymes Modification on Functional Properties of Soybean Protein Isolate Based on Orthogonal Experiment

Soybean protein isolate (SPI) is widely used in food industry because of its high protein nutritional function and good functional characteristics. However, due to the effect of amino acid composition and spatial structure on natural protein, its practical application is greatly limited. So it needs to be properly modified to meet the needs of production. In this study, SPI was used as substrate to explore the most suitable modification conditions by using complex enzymes (flavor protease, neutral protease, alkaline enzyme and transglutaminase) enzymolysis and then TG enzyme cross linking, in order to obtain SPI products with both solubility and gel as a special protein isolate for surimi products. The results show that: through the single factor experiment and orthogonal experiment, the optimized conditions of gel strength were determined: flavor protease: neutral protease: alkaline enzyme 1:1:2, pH 7, enzymolysis temperature 45°C, enzymolysis time 30 min. The optimized conditions of solubility: flavor protease: neutral protease: alkaline enzyme 1:2:2, pH 7, enzymolysis temperature 55°C, enzymolysis time 60 min. The result of orthogonal experiment: the optimized conditions was that flavor protease: neutral protease: alkaline enzyme 1:1:2, pH 7, enzymolysis temperature 55°C, enzymolysis time 60 min. The gel strength of products was 35.45 g, decreased 5.33% with control; Solubility was 36.24%, increased 54.01% with control. The modified SPI has excellent gel and solubility, and can be further applied to surimi products industry. And the results of this study provide a theoretical basis for its further application in surimi products.

Inhibitors of both the N -methyl lysyl- and arginyl-demethylase activities of the JmjC oxygenases

The Jumonji C (JmjC) family of 2-oxoglutarate (2OG)-dependent oxygenases have established roles in the regulation of transcription via the catalysis of demethylation of N ε - methylated lysine residues in histone tails, especially the N - terminal tail of histone H3. Most human JmjC N ɛ -methyl lysine demethylases (KDMs) are complex enzymes, with ‘reader domains’ in addition to their catalytic domains. Recent biochemical evidence has shown that some, but not all, JmjC KDMs also have N ω - methyl arginyl demethylase (RDM) activity. JmjC KDM activity has been linked to multiple cancers and some JmjC proteins are therapeutic targets. It is, therefore, important to test not only whether compounds in development inhibit the KDM activity of targeted JmjC demethylases, but also whether they inhibit other activities of these proteins. Here we report biochemical studies on the potential dual inhibition of JmjC KDM and RDM activities using a model JmjC demethylase, KDM4E (JMJD2E). The results reveal that all of the tested compounds inhibit both the KDM and RDM activities, raising questions about the in vivo effects of the inhibitors. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.