The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition.
Policy on Education is an important document for the nation. Education policy gives clear direction to the nation for achieving desired results. After Independence, Government has always strived to impart quality education for society without any discrimination. The New Education Policy, 2020 has been implemented after 34 years of the erstwhile Education Policy of 1986. The New Education Policy has overcome the drawbacks of previous ones and will establish global standards to meet the present as well as future requirements of the society. In this article, all these three policies are analysed with special reference to libraries. The New Education Policy will provide guidelines to build world class infrastructure to meet the demand of the users. More focus is given to digital libraries and also attention has been devoted to develop good collection of various discipline in regional languages also.
Vascular smooth muscle cells (SMCs) line blood vessels throughout the body, where they dynamically alter vessel diameter to regulate blood pressure, provide structural integrity, and absorb shock on a beat-to-beat timescale. As smooth muscle function fails, profound vascular disease ensues, often with tragic results- even death. Smooth muscle myosin 2 (SM2) is the dominant motor protein that actuates contractility and allows SMCs to perform these vital functions. To function, SM2 monomers dynamically assemble into filaments, which upon SMC activation, associate with filamentous actin to drive contractility. Despite the critical contribution of SM2 to SMC function, foundational aspects of SM2 assembly and dynamics remain unexplored. To remedy this, we expressed EGFP-tagged SM2 in rat aortic smooth muscle cells (A7R5), which retained a cytosolic calcium and contractile response to the acetylcholine agonist carbachol. Using fluorescence recovery after photobleaching (FRAP), we observed rapid polymer exchange kinetics for SM2, more similar to non-muscle myosin 2 (NM2) than striated myosin 2s. Consistently, super-resolution imaging of SM2 and NM2 suggests they form filamentous co-polymers. Using a single cell filament assembly assay, we observed that the majority of SM2 is assembled in filaments at steady-state, but that SMC activation with carbachol rapidly increases SM2 assembly levels. Carbachol also reduced polymer exchange kinetics, suggesting stabilization of filaments during SMC activation. This carbachol-induced increase in SM2 assembly and decrease in exchange kinetics closely parallels the cytosolic calcium and contractility kinetics. Collectively, our data supports an updated model in which highly dynamic SM2 filaments assemble, are stabilized, and are activated to produce cell-scale contractile forces during SMC activation.
The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.
ADP‐glucose pyrophosphorylase is the enzyme that controls the rate limiting step in the biosynthesis of glycogen and starch in bacteria and plants, respectively. In Agrobacterium tumefaciens, this enzyme is allosterically activated by fructose 6‐phosphate and pyruvate, whereas it is inhibited by AMP. In the crystal structure of the enzyme Ser72 interacts with a sulfate hypothesized to occupy the phosphate of the Fru6P allosteric site. To study the role of this residue, we replaced Ser72 by Ala, Cys, and Trp. In addition, we mutated it to Asp, and Glu to mimic the presence of negative charge of the Fru6P. Replacement by Ala did not significantly change the regulatory properties of the enzyme, indicating that the side chain does not play a critical role. However, we observed that S72D, S72E, S72C, and S72W became insensitive to Fru6P. Cys and Trp residues at position 72 remarkably reduced the specific activity of the enzyme (7.4 and 1.7 U/mg, respectively). On the other hand, S72D and S72E were partially activated (22.3 and 18.9 U/mg, respectively, compared to 8.3 U/mg of the WT). This indicates that the introduction of a carboxylate moiety in position 72 hinders the binding of Fru6P, but partially mimics its presence. These mutants were still activated by pyruvate, which indicates that this region is specifically involved in the activation by Fru6P and not by pyruvate. The mutants were not inhibited AMP inhibitor suggesting that the binding site for Fru6P and inhibitor overlaps. Our results suggest that Ser72 is in the Fru6P regulatory site and, consequently, the sulfate found in the crystal structure must be occupying the site of the phosphate moiety.Support or Funding InformationThis work was supported by NSF Grant MCB 1616851This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.