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New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

ABSTRACT: Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the 〈-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.

SUBMITTER: Selezneva AI

PROVIDER: S-EPMC10289334 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

Selezneva Anna I AI Harding Luke N M LNM Gutka Hiten J HJ Movahedzadeh Farahnaz F Abad-Zapatero Celerino C

PloS one 20230623 6

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product ...[more]

PMID: 37352301

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Project description:The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, β = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

Project description:Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel-Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.

Dataset Information

New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

Publications

New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

Similar Datasets

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