Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.2.13 (aldolase)
 3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 A) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na+ binding site is created using a pi interaction with Tyr183 in addition to five oxygen ligands. The catalytic Zn2+ is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry.
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PMID:Structure of tagatose-1,6-bisphosphate aldolase. Insight into chiral discrimination, mechanism, and specificity of class II aldolases. 1194 Jun 3

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.
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PMID:Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa. 1223 94

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.
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PMID:Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization). 1223 96

Full details of our newly developed catalyses with asymmetric zinc complexes as mimics of class II zinc-containing aldolase are described. A Et(2)Zn/(S,S)-linked-BINOL complex was developed and successfully applied to direct catalytic asymmetric aldol reactions of hydroxyketones. A Et(2)Zn/(S,S)-linked-BINOL 1 = 2/1 system was initially developed, which efficiently promoted the direct aldol reaction of 2-hydroxy-2'-methoxyacetophenone (7d). Using 1 mol % of (S,S)-linked-BINOL 1 and 2 mol % of Et(2)Zn, we obtained 1,2-dihydroxyketones syn-selectively in high yield (up to 95%), good diastereomeric ratio (up to 97/3), and excellent enantiomeric excess (up to 99%). Mechanistic investigation of Et(2)Zn/(S,S)-linked-BINOL 1, including X-ray analysis, NMR analysis, cold spray ionization mass spectrometry (CSI-MS) analysis, and kinetic studies, provided new insight into the active oligomeric Zn/(S,S)-linked-BINOL 1/ketone 7d active species. On the basis of mechanistic investigations, a modified second generation Et(2)Zn/(S,S)-linked-BINOL 1 = 4/1 with molecular sieves 3A (MS 3A) system was developed as a much more effective catalyst system for the direct aldol reaction. As little as 0.1 mol % of (S,S)-linked-BINOL 1 and 0.4 mol % of Et(2)Zn promoted the direct aldol reaction smoothly, using only 1.1 equiv of 7d as a donor (substrate/ligand = 1000). This is the most efficient, in terms of catalyst loading, asymmetric catalyst for the direct catalytic asymmetric aldol reaction. Moreover, the Et(2)Zn/(S,S)-linked-BINOL 1 = 4/1 system was effective in the direct catalytic asymmetric aldol reaction of 2-hydroxy-2'-methoxypropiophenone (12), which afforded a chiral tetrasubstituted carbon center (tert-alcohol) in good yield (up to 97%) and ee (up to 97%), albeit in modest syn-selectivity. Newly developed (S,S)-sulfur-linked-BINOL 2 was also effective in the direct aldol reaction of 12. The Et(2)Zn/(S,S)-sulfur-linked-BINOL 2 = 4/1 system gave aldol adducts anti-selectively in good ee (up to 93%). Transformations of the aldol adducts into synthetically versatile intermediates were also described.
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PMID:Direct catalytic asymmetric aldol reaction of hydroxyketones: asymmetric Zn catalysis with a Et(2)Zn/linked-BINOL complex. 1259 May 45

Previously determined crystal structures of the zinc enzyme Escherichia coli class II fructose-1,6-bisphosphate aldolase display good agreement for the protein structure but a differing metal-ion organization in the active site. The structure of the enzyme with Cd(2+) in place of Zn(2+) has now been determined to 2.0 A resolution to facilitate cation identification. The protein structure was essentially identical to other structures and five Cd(2+) positions were identified. Two of the cations are at the active site; one corresponds to the catalytic ion and the other provides a structural contribution. These Cd(2+) sites are equivalent to two Zn(2+) ions observed when the enzyme is complexed with a transition-state mimic and confirm our assignment of the roles played by these ions.
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PMID:The organization of divalent cations in the active site of cadmium Escherichia coli fructose-1,6-bisphosphate aldolase. 1259 41

The structure of L-rhamnulose-1-phosphate aldolase has been established at 1.35 A resolution in a crystal form that was obtained by a surface mutation and has one subunit of the C(4)-symmetric tetramer in the asymmetric unit. It confirms an earlier 2.7 A resolution structure which was determined in a complicated crystal form with 20 subunits per asymmetric unit. The chain fold and the active center are similar to those of L-fuculose-1-phosphate aldolase and L-ribulose-5-phosphate 4-epimerase. The active center similarity is supported by a structural comparison of all three enzymes and by the binding mode of the inhibitor phosphoglycolohydroxamate at the site of the product dihydroxyacetone phosphate for the two aldolases. The sensitivity of the catalytic rate to several mutations and a comparison with the established mechanism of the related aldolase give rise to a putative catalytic mechanism. This mechanism involves the same binding mode of the second product L-lactaldehyde in both aldolases, except for a 180 degrees flip of the aldehyde group distinguishing between the two epimers rhamnulose and fuculose. The N-terminal domain exhibits a correlated anisotropic mobility that channels the isotropic Brownian motion into a directed movement of the catalytic base and the substrate phosphate on the N-domain toward the zinc ion and the lactaldehyde on the C-terminal domain. We suggest that this movement supports the catalysis mechanically.
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PMID:Structure and catalytic mechanism of L-rhamnulose-1-phosphate aldolase. 1296 79

The leucine biosynthetic pathway is essential for the growth of Mycobacterium tuberculosis and is a potential target for the design of new anti-tuberculosis drugs. The crystal structure of alpha-isopropylmalate synthase, which catalyzes the first committed step in this pathway, has been determined by multiwavelength anomalous dispersion methods and refined at 2.0-A resolution in complex with its substrate alpha-ketoisovalerate. The structure reveals a tightly associated, domain-swapped dimer in which each monomer comprises an (alpha/beta)(8) TIM barrel catalytic domain, a helical linker domain, and a regulatory domain of novel fold. Mutational and crystallographic data indicate the latter as the site for leucine feedback inhibition of activity. Domain swapping enables the linker domain of one monomer to sit over the catalytic domain of the other, inserting residues into the active site that may be important in catalysis. The alpha-ketoisovalerate substrate binds to an active site zinc ion, adjacent to a cavity that can accommodate acetyl-CoA. Sequence and structural similarities point to a catalytic mechanism similar to that of malate synthase and an evolutionary relationship with an aldolase that catalyzes the reverse reaction on a similar substrate.
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PMID:Crystal structure of LeuA from Mycobacterium tuberculosis, a key enzyme in leucine biosynthesis. 1515 44

The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.
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PMID:Molecular cloning, expression, purification, and characterization of fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis--a novel Class II A tetramer. 1529 2

The structure of the class II zinc-ion dependent L-fuculose-1-phosphate aldolase from Escherichia coli in its tetragonal crystal form has been established at 1.92 A resolution. The homotetrameric enzyme has a molecular mass of 4 x 24 kDa and follows C(4) symmetry. The structure model is exactly symmetrical, which contradicts an observed birefringence anomaly of the crystals. The four catalytic centers are located in deep clefts at the interfaces of adjacent subunits. The zinc ion is coordinated by three histidines and one glutamate in an almost tetrahedral arrangement. In contrast to numerous other catalytically competent zinc ions, there is no water molecule in the ligand sphere. Replacement of zinc by a cobalt ion caused only small structural changes. A search through the Protein Data Bank indicated that the chain fold is novel. Sequence homology searches revealed a significant similarity to the bacterial L-ribulose-5-phosphate 4-epimerase.
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PMID:Refined high-resolution structure of the metal-ion dependent L-fuculose-1-phosphate aldolase (class II) from Escherichia coli. 1529 67

Fuculose phosphate aldolase catalyzes the reversible cleavage of L-fuculose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 A. The presence of a dimer of the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (VM) of 2.7 A3 Da(-1) and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 A from zinc-containing crystals. Native diffraction data to 1.9 A resolution have been collected using synchrotron radiation at SPring-8.
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PMID:Purification, crystallization and preliminary X-ray crystallographic study of the L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8. 1651 Dec 38


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