Dual role of strigolactone receptor signaling partner in inhibiting substrate hydrolysis

Plant branch and root growth relies on metabolism of the strigolactone (SL) hormone. The interaction between the SL molecule, Oryza sativa DWARF14 (D14) SL receptor, and D3 F-box protein has been shown to play a critical role in SL perception. Previously, it was believed that D3 only interacts with the closed form of D14 to induce downstream signaling, but recent experiments indicate that D3, as well as its C-terminal helix (CTH), can interact with the open form as well to inhibit strigolactone signaling. Two hypotheses for the CTH induced inhibition are that either the CTH affects the conformational ensemble of D14 by stabilizing catalytically inactive states, or the CTH interacts with SLs in a way that prevents them from entering the binding pocket. In this study, we have performed molecular dynamics (MD) simulations to assess the validity of these hypotheses. We used an apo system with only D14 and the CTH to test the active site conformational stability and a holo system with D14, the CTH, and an SL molecule to test the interaction between the SL and CTH. Our simulations show that the CTH affects both active site conformation and the ability of SLs to move into the binding pocket. In the apo system, the CTH allosterically stabilized catalytic residues into their inactive conformation. In the holo system, significant interactions between SLs and the CTH hindered the ability of SLs to enter the D14 binding pocket. These two mechanisms account for the observed decrease in SL binding to D14 and subsequent ligand hydrolysis in the presence of the CTH.

Opening the side exit pores of ClpP by lowering the pH of proteolytic chamber coupled with substrate hydrolysis

The ClpP serine peptidase is a tetradecameric degradation machine involved in many physiological processes. It becomes a competent ATP-dependent protease with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause dysregulation and activation of ClpP without ATPases, and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed the structural details, conformational changes, and activation mechanism. Although product release by the side exit pores has been proposed, the detailed driving force for product release remains elusive. Here, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures revealed various conformational states at different pH conditions. To understand the conformational change for product release, we investigated the relationship between substrate hydrolysis and the pH lowering process. Our data, together with previous findings, provide insight into the molecular mechanism of product release by ClpP self-compartmentalizing protease.

Description Of Associative Behaviour During Microbial Cellulose Utilization By Clostridium Thermocellum For Application In Advanced Biomass Conversion Process Development

Recent findings support the concept that Clostridium thermocellum is a cellulose-utilizing specialist having growth benefits with increasing substrate chain length. We developed a continuous-flow system for in-situ detection of cellulose colonization and qualitatively assayed metabolic activities and behaviour of cellulolytic cultures. This study demonstrates the existence of strongly adherent celluloytic cells arranged in monolayers with invariably end-on attached spores. The substrate-cell distance was recorded to be lower than 0.44 pm and a typical EPS matrix was absent. Measurements on carbon dioxide released in continuous-flow cultures was successfully employed to monitor biofilm activity and total carbohydrate assays do not reveal loss of cellulolysis end-products in the effluent. These findings demonstrate the bacteria have optimized access to the cellulosic substrates and suggest that they have an ability to sequester products of substrate hydrolysis which confers benefits over non-adherent cellulolytic or non-cellulolytic organisms.

Description Of Associative Behaviour During Microbial Cellulose Utilization By Clostridium Thermocellum For Application In Advanced Biomass Conversion Process Development

Recent findings support the concept that Clostridium thermocellum is a cellulose-utilizing specialist having growth benefits with increasing substrate chain length. We developed a continuous-flow system for in-situ detection of cellulose colonization and qualitatively assayed metabolic activities and behaviour of cellulolytic cultures. This study demonstrates the existence of strongly adherent celluloytic cells arranged in monolayers with invariably end-on attached spores. The substrate-cell distance was recorded to be lower than 0.44 pm and a typical EPS matrix was absent. Measurements on carbon dioxide released in continuous-flow cultures was successfully employed to monitor biofilm activity and total carbohydrate assays do not reveal loss of cellulolysis end-products in the effluent. These findings demonstrate the bacteria have optimized access to the cellulosic substrates and suggest that they have an ability to sequester products of substrate hydrolysis which confers benefits over non-adherent cellulolytic or non-cellulolytic organisms.

Enzyme Kinetics Study for Heterogeneous System of Pretreated Kenaf Hydrolysis

The peculiarity of spatially restricted diffusion and molecular collision processes results in considerable contrast in a reaction between the reactant and catalyst in the heterogeneous system from its corresponding homogeneous structure. The identification of the enzymatic hydrolysis process of pre-treated kenaf and to convert it into simple sugars employing a systematic kinetic investigation is the aims of this study. The influence of substrate concentration on xylanase hydrolysis was performed in water bath shakers. In-house recombinant xylanase expressed in Pichia pastoris was used for the hydrolysis at pH 4.0 in 50 mM sodium citrate buffer with 200 rpm agitation. Modified Prout-Tompkins equation was used for the heterogeneous substrate hydrolysis. The results obtained show that temperature simultaneously influenced the time dependency of the reducing sugar yield. Dependence of the enzymatic rate of reaction can be calculated effectively on the conversion of substrates over different temperatures. The activation energy needed for pretreated kenaf hydrolysis was among the least compared to other lignocelluloses, which was only 25.15 kJ/mol. In conclusion, the exponential kinetic equation by the Modified Prout-Tompkins equation offers a solid understanding of xylanase hydrolysis on the pretreated kenaf. Thus, the prediction of the degree of hydrolysis required at the predetermined temperature and time values used can be quickly and precisely determined.

Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-d-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). Conclusions Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural “key code” present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

Background: For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes; with the optimum combined activity ratio dependent on the composition of the substrate. Results: In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2 respectively (ratio of 1.6:1). Conclusions: Here we describe a synthetic biology approach to rapidly optimize gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

Determination of Kinetic Parameters and The Effect of Ion Mg2+ Inhibition Into Pectinase Activities.

The purpose of study was to determine kinetic parameters and inhibitory effect of Mg2+ ions on pectinase. Activity test performed at pH 4,5 temperature 55oC for 50' with 10 mm concentrations of each ion 0-10mm. Galacturonic acid content, as product of pectin substrate hydrolysis, was used as basis for determining activity and analyzed by visible spectrophotometry. At concentrations 2 and 4 mm Mg2+ act as an activator, but at concentrations 6, 8, and 10 mM as inhibitors. The value of KM with and without inhibitors is almost same, that is 0.3145% and 0.3105% but value of Vm from both are different, that is 80,645 μg.ml-1.minute-1 and 62,112 μg.ml-1.minute-1. The conclusion that type of inhibitory pectinase with Mg2+ is a non-competitive inhibition. The inhibition constant value (Ki) is 26,84.