Carbohydrate response element binding protein, ChREBP, a transcription factor coupling hepatic glucose utilization and lipid synthesis

The ability of an organism to sense and store nutrients is vital to survival. The liver is the major organ responsible for converting excess dietary carbohydrate to lipid for storage. An elegant molecular pathway has evolved that allows increased glucose flux into hepatocytes to generate a signaling molecule, xylulose 5-phosphate, that triggers rapid changes in glycolytic enzyme activities and nuclear import of a transcription factor, ChREBP, which coordinates the transcriptional regulation of enzymes that channel the glycolytic end-products into lipogenesis. Further understanding of this metabolic cascade should provide insights on conditions such as fatty liver, obesity, and the metabolic syndrome

Tissue-specific structure/function differentiation of the liver isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The crystal structures of the human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in three different liganding states were determined and compared with those of the rat testis isozyme. A set of amino acid sequence heterogeneity from the two distinct genes encoding the two different tissue isozymes leads to both global and local conformational differences that may cause the differences in catalytic properties of the two isozymes. The sequence differences in a β-hairpin loop in the kinase domain causes a translational shift of several hydrophobic interactions in the dimeric contact region, and its propagation to the domains interface results in a 5° twist of the entire bisphosphatase domain relative to the kinase domain. The bisphosphatase domain twist allows the dimeric interactions between the bisphosphatase domains, which are negligible in the testis enzyme, and as a result, the conformational stability of the domain is increased. Sequence polymorphisms also confer small but significant structural dissimilarities in the substrate-binding loops, allowing the differentiated catalytic properties between the two different tissue-type isozymes. Whereas the polymorphic sequence at the bisphosphatase-active pocket suggests a more suitable substrate binding, a similar extent of sequence differences at the kinase-active pocket confers a different mechanism of substrates bindings to the kinase-active pocket. It includes the ATP-sensitive unwinding of the switch helix α5, which is a characteristic ATP-dependent conformational change in the testis form. The sequence-dependent structural difference disallows the liver kinase to follow the ATP-switch mechanism. Altogether these suggest that the liver isoform has structural features more appropriate for an elevated bisphosphatase activity, compared with that of the testis form. The structural predisposition for bisphosphatase activity in the liver isozyme is consistent with the liver-unique glucose metabolic pathway, gluconeogenesis

Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase: Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2- kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of β-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru- 2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166- 2175). We have solved the crystal structure of H256A to a resolution of 2.4 Å by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes

The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies

AbstractBackground Glucose homeostasis is maintained by the processes of glycolysis and gluconeogenesis. The importance of these pathways is demonstrated by the severe and life threatening effects observed in various forms of diabetes. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Thus this bifunctional enzyme plays an indirect yet key role in the regulation of glucose metabolism.Results We have determined the 2.0 å crystal structure of the rat testis isozyme of this bifunctional enzyme. The enzyme is a homodimer of 55 kDa subunits arranged in a head-to-head fashion, with each monomer consisting of independent kinase and phosphatase domains. The location of ATPγS and inorganic phosphate in the kinase and phosphatase domains, respectively, allow us to locate and describe the active sites of both domains.Conclusions The kinase domain is clearly related to the superfamily of mononucleotide binding proteins, with a particularly close relationship to the adenylate kinases and the nucleotide-binding portion of the G proteins. This is in disagreement with the broad speculation that this domain would resemble phosphofructokinase. The phosphatase domain is structurally related to a family of proteins which includes the cofactor independent phosphoglycerate mutases and acid phosphatases

Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA

This study demonstrates the bottom-up creation of an artificial cell which carries an active enzyme inside the cell and exhibit adhesive function to both hydrophobic and hydrophilic surfaces using AtaA, the adhesive bacterionanofiber protein from Acinetobacter sp. Tol 5, chemically coupled to the lipid membrane of the artificial cell