Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.     

Broad substrate stereospecificity of the Mycobacterium tuberculosis 7-keto-8-aminopelargonic acid synthase: Spectroscopic and kinetic studies

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the mycobacterium tuberculosis KAPA synthase with both L- AND D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 m(-1) s(-1)) is approximately 5 times slower than that with L-alanine (k = 399.4 m(-1) s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed d-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 10(4) m(-1) s(-1)). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, d-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (Ki = 114.83 microm) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 microm), the next enzyme of the pathway.     

Crystallization and preliminary crystallographic studies of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli

3-Deoxy-D-manno-octulosonate-8-phosphate (KDOP) synthase catalyzes the production of KDOP from phosphoenolpyruvate (PEP) and arabinose-5-phosphate (A5P). In gram-negative bacteria KDOP is subsequently dephosphorylated, cytidylylated, and linked to lipid A and is required for lipid A incorporation into the outer membrane (Raetz, Annu. Rev. Biochem. 59:129–170, 1990). We have crystallized two forms of KDOP synthase belonging to space groups I23 or I2₁3, one with a = b = c = 118.0 Å and the other with a = b = c = 233 Å.     

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.     

NMR spectroscopic, mass spectroscopic, X-ray crystallographic, and theoretical studies of molecular mechanics of natural products: farformolide B and sesamin

Two natural products, farformolide B and sesamin were isolated from Farfugium japonicum and Cinnamomum kanehirae, respectively. The structures of the two natural products, including their relative stereochemistry, were elucidated using spectroscopic data and theoretical calculations. The molecule 1 (farformolide B) is newly recognized by X-ray crystallography. The two compounds were also investigated by a theoretical analysis using the B3LYP/6-31G* method of the Gaussian 03 package program. The theoretical results were supplemented by experimental data to determine the optimal geometric structures of the two compounds. The calculated molecular mechanics were found to compare well with the experimental data. Several important thermodynamic properties of the two products, including ionization potentials, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies, energy gaps, heat of formation, atomization energies, and vibration frequencies, were also calculated. The study also provided a good understanding of the stereochemical structure and thermodynamic properties of the two molecules.     

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.     

Polymorphism of N-stearoylethanolamine: differential scanning calorimetric,vibrational spectroscopic (FTIR), and crystallographic studies

Based on a series of physicochemical properties (differential scanning calorimetry, powder X-ray crystallographic studies and Fourier-transform infra red spectroscopic analysis) determined for N-stearoylethanolamine (NSEA) (C18:0) at different temperatures, evidence has been given that this compound can exist in (at least) three polymorphic forms. Powder X-ray crystallography clearly demonstrates the presence of three distinct molecular packings at distinct temperatures while spectral changes in the vibrational spectra reveal that the geometry of the CH(2)z.sbnd;CO functional group of the molecule is affected during the polymorphic transitions. Rationalization of the thermal physicochemical behavior of NSEA in terms of molecular packing is also proposed. It supposes rearrangement of the hydrocarbon chains upon heating of the molecule.     

Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.     

Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.     

The binding of beta- and gamma-cyclodextrins to glycogen phosphorylase b: kinetic and crystallographic studies

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that beta- and gamma-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of alpha-cyclodextrin and octakis (2,3,6-tri-O-methyl)-gamma-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.     

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis.     

Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: crystallographic, mutagenesis and resonance Raman spectroscopic studies

FixL of Rhizobium meliloti (RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O(2) levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 A resolution. Based on the structural and spectroscopic analyses, we propose the O(2) sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O(2) and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O(2) and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O(2) and Fe-CN stretching frequencies were shifted from 575 to 570 cm(-1) (Fe-O(2)), and 504 to 499 cm(-1), respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O(2) sensing activity was destroyed, thus confirming our proposed mechanism.     

Mechanism of Agrobacterium beta-glucosidase: kinetic studies.

The beta-glucosidase from Agrobacterium faecalis (previously Alcaligenes faecalis) has been subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It has a relatively broad specificity for the substrate sugar moiety and exhibits a classical pH dependence for its kinetic parameters with three different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates has allowed construction of a Bronsted plot, the concave-downward shape of which is consistent with the anticipated two-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Bronsted plot (beta 1g = -0.7) indicates a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for five different aryl glucosides are also consistent with this mechanism and further suggest that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD = 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analogue inhibitors are presented which support the concept of sp2-hybridized transition states.     

Mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase: kinetic studies

The catalytic mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) from family 39 of glycoside hydrolases has been subjected to a detailed kinetic investigation using a range of substrates. The enzyme exhibits a bell-shaped pH dependence of k(cat)/K(m), reflecting apparent pK(a) values of 4.1 and 6.8. The k(cat) and k(cat)/K(m) values for a series of aryl xylosides have been measured and used to construct two Br?nsted plots. The plot of log(k(cat)/K(m)) against the pK(a) of the leaving group reveals a significant correlation (beta(lg) = -0.97, r(2) = 0.94, n = 8), indicating that fission of the glycosidic bond is significantly advanced in the transition state leading to the formation of the xylosyl-enzyme intermediate. The large negative value of the slope indicates that there is relatively little proton donation to the glycosidic oxygen in the transition state. A biphasic, concave-downward plot of log(k(cat)) against pK(a) provides good evidence for a two-step double-displacement mechanism involving a glycosyl-enzyme intermediate. For activated leaving groups (pK(a) < 9), the breakdown of the xylosyl-enzyme intermediate is the rate-determining step, as indicated by the absence of any effect of the pK(a) of the leaving group on log(k(cat)) (beta(lg) approximately 0). However, a strong dependence of the first-order rate constant on the pK(a) value of relatively poor leaving groups (pK(a) > 9) suggests that the xylosylation step is rate-determining for these substrates. Support for the dexylosylation chemical step being rate-determining for activated substrates comes from nucleophilic competition experiments in which addition of dithiothreitol results in an increase in turnover rates. Normal secondary alpha-deuterium kinetic isotope effects ((alpha-D)(V) or (alpha-D)(V/K) = 1.08-1.10) for three different substrates of widely varying pK(a) value (5.15-9.95) have been measured and these reveal that the transition states leading to the formation and breakdown of the intermediate are similar and both steps involve rehybridization of C1 from sp(3) to sp(2). These results are consistent only with "exploded" transition states, in which the saccharide moiety bears considerable positive charge, and the intermediate is a covalent acylal-ester where C1 is sp(3) hybridized.     

Purification,crystallization, and preliminary crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

The phenylalanine-regulated isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate- synthase (DAHPS) from Escherichia coli, its binary complexes with either substrate, phosphoenolpyruvate (PEP), or feedback inhibitor, Phe, and its ternary complexes with either PEP or Phe plus metal cofactor (either Mn²⁺, Cd²⁺, or Pb²⁺) were crystallized from polyethylglycol (PEG) solutions. All crystals of the DAHPS without Phe belong to space group C2, with cell parameters a = 213.5 Å, b = 54.3 Å, c = 149.0 Å, β = 116.6°. All crystals of the enzyme with Phe also belong to space group C2, but with cell parameters a = 297.1 Å, b = 91.4 Å, c = 256.5 Å, and β = 148.2°.     

Physicochemical characterization of deflazacort: thermal analysis, crystallographic and spectroscopic study

The solid state properties of deflazacort (1-(1beta,16alpha)-21-(acetyloxy)-11-hydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione, 1) were investigated using differential scanning calorimetry (DSC), thermogravimetry (TG), single-crystal X-ray diffraction, solid and liquid nuclear magnetic resonance spectroscopy ((13)C NMR), Fourier transform infrared and Raman spectroscopy (FTIR and FT Raman). From the trends observed in the crystal structure and spectral data some conclusions can be made about hydrogen bonding, molecular conformation and crystal packing. Compound 1 crystallizes in an orthorhombic cell, space group P2(1)2(1)2(1) and the following lattice parameters: a=11.2300(5), b=12.8161(8), c=16.171(1)A, V: 2327.4(2)A(3), D(c): 1.260g/cm(3), R1=0.0479, wR2=0.1012. The crystal structure is stabilized by intra and intermolecular interactions, which provides for a very closely packed form. The NMR data indicated that 1 shows a similar conformation in solid and liquid state; while, thermal data revealed that 1 follows a monophasic pattern with a DSC melting peak at 258.4 degrees C (DeltaH 99.7Jg(-1), n=3), indicating that 1 is thermally stable as solid; but, as liquid is unstable to undergo a thermal decomposition reaction. The reactivity of 1 toward light and moisture was examined via DSC and TLC. The data indicated that 1 do not interact with water to give hydrated forms or decomposition products; however, light degrades 1.     

Transient kinetic studies support refinements to the chemical and kinetic mechanisms of aminolevulinate synthase

5-Aminolevulinate synthase catalyzes the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate alpha-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 degrees C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pK(a) of 7.7 +/- 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the alpha-amino-beta-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pK(a) of 8.1 +/- 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines k(cat) and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis.