scholarly journals Structures of theMycobacterium tuberculosisGlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

2018 ◽  
Vol 74 (4) ◽  
pp. 321-331
Author(s):  
Nina M. Wolf ◽  
Hiten J. Gutka ◽  
Farahnaz Movahedzadeh ◽  
Celerino Abad-Zapatero
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Adenosine Monophosphate ◽  
Class Ii ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Oligomeric State ◽  
Fructose 1,6 Bisphosphatase ◽  
Lead Inhibitors

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) fromMycobacterium tuberculosisat 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase fromSynechocystis(strain 6803) as well as the equivalent enzyme fromThermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII fromEscherichia coliand is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII fromM. tuberculosis(MtFBPaseII) is conserved and is analogous to that described previously for theE. colienzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures ofMtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for theSynechocystisenzyme. The structural and functional insights derived from the structure ofMtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals T-to-R switch of muscle fructose-1,6-bisphosphatase involves fundamental changes of secondary and quaternary structure

2016 ◽  
Vol 72 (4) ◽  
pp. 536-550 ◽  
Author(s):  
Jakub Barciszewski ◽  
Janusz Wisniewski ◽  
Robert Kolodziejczyk ◽  
Mariusz Jaskolski ◽  
Dariusz Rakus ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Homo Sapiens ◽  
Helical Structure ◽  
Smooth Transition ◽  
Muscle Enzyme ◽  
Enzymatic Function ◽  
Fructose 1,6 Bisphosphatase ◽  
Key Enzyme

Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and is a key enzyme of gluconeogenesis and glyconeogenesis and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrates, and notablyHomo sapiens, express two FBPase isoforms. The liver isozyme is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis. The muscle isoform is expressed in all cells, and recent studies have demonstrated that its role goes far beyond the enzymatic function, as it can interact with various nuclear and mitochondrial proteins. Even in its enzymatic function, the muscle enzyme is different from the liver isoform, as it is 100-fold more susceptible to allosteric inhibition by AMP and this effect can be abrogated by complex formation with aldolase. All FBPases are homotetramers composed of two intimate dimers: the upper dimer and the lower dimer. They oscillate between two conformational states: the inactive T form when in complex with AMP, and the active R form. Parenthetically, it is noted that bacterial FBPases behave somewhat differently, and in the absence of allosteric activators exist in a tetramer–dimer equilibrium even at relatively high concentrations. [Hineset al.(2007),J. Biol. Chem.282, 11696–11704]. The T-to-R transition is correlated with the conformation of the key loop L2, which in the T form becomes `disengaged' and unable to participate in the catalytic mechanism. The T states of both isoforms are very similar, with a small twist of the upper dimer relative to the lower dimer. It is shown that at variance with the well studied R form of the liver enzyme, which is flat, the R form of the muscle enzyme is diametrically different, with a perpendicular orientation of the upper and lower dimers. The crystal structure of the muscle-isozyme R form shows that in this arrangement of the tetramer completely new protein surfaces are exposed that are most likely targets for the interactions with various cellular and enzymatic partners. The cruciform R structure is stabilized by a novel `leucine lock', which prevents the key residue, Asp187, from locking loop L2 in the disengaged conformation. In addition, the crystal structures of muscle FBPase in the T conformation with and without AMP strongly suggest that the T-to-R transition is a discrete jump rather than a shift of an equilibrium smooth transition through multiple intermediate states. Finally, using snapshots from three crystal structures of human muscle FBPase, it is conclusively demonstrated that the AMP-binding event is correlated with a β→α transition at the N-terminus of the protein and with the formation of a new helical structure.

scholarly journals Regulatory Properties of Apple Leaf Cytosolic Fructose-1,6-bisphosphatase

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 761C-761
Author(s):  
Rui Zhou* ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Adenosine Monophosphate ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Carbon Partitioning ◽  
Dihydroxyacetone Phosphate ◽  
Saturation Curve ◽  
Fruit Species ◽  
Apparent Homogeneity ◽  
Fructose 1,6 Bisphosphatase

Cytosolic fructose-1,6-bisphosphatase (cytoFBPase) (EC 3.1.3.11) occupies a strategic site in sucrose synthesis and has been demonstrated to play a key role in carbon partitioning between sucrose and starch in non-sorbitol forming plants. In addition to sucrose and starch, Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family. To understand the biochemical regulation of photosynthetic carbon partitioning between sorbitol, sucrose and starch in sorbitol synthesizing species, we purified cytoFB-Pase to apparent homogeneity from apple leaves. The enzyme was a homotetramer with a subunit mass of 37 kDa. It was highly specific for fructose-1,6-bisphosphate with a Km of 3.1 μm and a Vmax of 48 units/mg protein. Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mm and 62 μM, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+-activated enzyme activity. Fructose-6-phosphate was found to be a mixed type inhibitor with a Ki of 0.47 mm. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. Adenosine monophosphate (AMP) was a non-competitive inhibitor for the enzyme. F2,6BP interacted with AMP to inhibit the enzyme in a synergistic way. Dihydroxyacetone phosphate did not have inhibitory effect on apple leaf cytosolic FBPase activity. Sorbitol increased the susceptibility of the enzyme to the inhibition by F1,6BP. The presence of sorbitol in the reaction mixture led to a reduction in the enzyme activity.

scholarly journals Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis

2020 ◽  
Vol 76 (11) ◽  
pp. 524-535
Author(s):  
Anna I. Selezneva ◽  
Hiten J. Gutka ◽  
Nina M. Wolf ◽  
Fnu Qurratulain ◽  
Farahnaz Movahedzadeh ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Active Site ◽  
Biochemical Characterization ◽  
Structural Characteristics ◽  
Crystal Form ◽  
Class Ii ◽  
Monoclinic Crystal ◽  
Cell Parameters ◽  
Functional Relationships ◽  
Fructose 1,6 Bisphosphatase

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 (D 2) 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 K m and V max 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.

scholarly journals Synthesis of 2-(4-hydroxyphenyl)ethyl 3,4,5-Trihydroxybenzoate and Its Inhibitory Effect on Sucrase and Maltase

Processes ◽  
2020 ◽  
Vol 8 (12) ◽  
pp. 1603
Author(s):  
Wen-Tai Li ◽  
Yu-Hsuan Chuang ◽  
Jiahn-Haur Liao ◽  
Jung-Feng Hsieh
Keyword(s):  
Active Site ◽  
Docking Study ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Kinetics Study ◽  
Amino Acid Residues ◽  
Inhibition Kinetics ◽  
Molecular Docking Study ◽  
Hydrogen Bond Interactions ◽  
Glucosidase Inhibitors

We report on the synthesis of an active component, 2-(4-hydroxyphenyl)ethyl 3,4,5-trihydroxybenzoate (HETB), from Rhodiola crenulata. Subsequent analysis revealed that HETB exhibits α-glucosidase inhibitory activities on maltase and sucrase, with potency exceeding that of the known α-glucosidase inhibitors (voglibose and acarbose). An inhibition kinetics study revealed that HETB, acarbose, and voglibose bind to maltase and sucrase, and HETB was shown to be a strong competitive inhibitor of maltase and sucrase. In a molecular docking study based on the crystal structure of α-glucosidase from Saccharomyces cerevisiae, we revealed the HETB binding in the active site of maltase via hydrogen-bond interactions with five amino acid residues: Ser 240, Asp 242, Glu 277, Arg 315, and Asn 350. For HETB docked to the sucrase active site, seven hydrogen bonds (with Asn 114, Glu 148, Gln 201, Asn 228, Gln 381, Ile 383, and Ser 412) were shown.

scholarly journals Structure of Allium lachrymatory factor synthase elucidates catalysis on sulfenic acid substrate

2017 ◽  
Author(s):  
Takatoshi Arakawa ◽  
Yuta Sato ◽  
Jumpei Takabe ◽  
Noriya Masamura ◽  
Masahiro Kato ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Sigmatropic Rearrangement ◽  
Natural Production ◽  
Lachrymatory Factor ◽  
Biological Decomposition ◽  
Key Residues ◽  
Lachrymatory Factor Synthase

AbstractNatural lachrymatory effects are invoked by small volatile S-oxide compounds. They are produced through alkene sulfenic acids by the action of lachrymatory factor synthase (LFS). Here we present the crystal structures of onion LFS (AcLFS) revealed in solute-free and two solute-stabilized forms. Each structure adopts a single seven-stranded helix-grip fold possessing an internal pocket. Mutagenesis analysis localized the active site to a layer near the bottom of the pocket, which is adjacent to the deduced key residues Arg71, Glu88, and Tyr114. Solute molecules visible on the active site have suggested that AcLFS accepts various small alcohol compounds as well as its natural substrate, and they inhibit this substrate according to their chemistry. Structural homologs have been found in the SRPBCC superfamily, and comparison of the active sites has demonstrated that the electrostatic potential unique to AcLFS could work in capturing the substrate in its specific state. Finally, we propose a rational catalytic mechanism based on intramolecular proton shuttling in which the microenvironment of AcLFS can bypass the canonical [1,4]-sigmatropic rearrangement principle established by microwave studies. Beyond revealing how AcLFS generates the lachrymatory compound, this study provides insights into the molecular machinery dealing with highly labile organosulfur species.Significance statementCrushing of onion liberates a volatile compound, syn-propanethial S-oxide (PTSO), which causes lachrymatory effect on humans. We present the crystal structures of onion LFS (AcLFS), the enzyme responsible for natural production of PTSO. AcLFS features a barrel-like fold, and mutagenic and inhibitory analyses revealed that the key residues are present in the central pocket, harboring highly concentrated aromatic residues plus a dyad motif. The architecture of AcLFS is widespread among proteins with various biological functions, such as abscisic acid receptors and polyketide cyclases, and comparisons with these homologs indicate that unique steric and electronic properties maintain the pocket as a reaction compartment. We propose the molecular mechanism behind PTSO generation and shed light on biological decomposition of short-lived sulfur species.

scholarly journals Evidence for essential histidine and dicarboxylic amino-acid residues in the active site of UDP-glucose : solasodine glucosyltransferase from eggplant leaves.

2003 ◽  
Vol 50 (2) ◽  
pp. 567-572 ◽  
Author(s):  
Paulina Nawłoka ◽  
Małgorzata Kalinowska ◽  
Cezary Paczkowski ◽  
Zdzisław A Wojciechowski
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Active Site ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Chemical Probes ◽  
Dicarboxylic Amino Acid ◽  
Enzyme Substrates ◽  
Arginine Residues

Effects of several chemical probes selectively modifying various amino-acid residues on the activity of UDP-glucose : solasodine glucosyltransferase from eggplant leaves was studied. It was shown that diethylpyrocarbonate (DEPC), a specific modifier of histidine residues, was strongly inhibitory. However, in the presence of excessive amounts of the enzyme substrates, i.e. either UDP-glucose or solasodine, the inhibitory effect of DEPC was much weaker indicating that histidine (or histidines) are present in the active site of the enzyme. Our results suggest also that unmodified residues of glutamic (or aspartic) acid, lysine, cysteine, tyrosine and tryptophan are necessary for full activity of the enzyme. Reagents modifying serine and arginine residues have no effect on the enzyme activity.

scholarly journals Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Martino L. Di Salvo ◽  
J. Neel Scarsdale ◽  
Galina Kazanina ◽  
Roberto Contestabile ◽  
Verne Schirch ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Serine Hydroxymethyltransferase ◽  
Active Site Residue ◽  
Wild Type ◽  
Hydroxymethyl Group ◽  
New Energy ◽  
Mutant Forms

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stablegem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion ofgem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and theε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

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