Search

Pantothenate synthetase (PS) from Mycobacterium tuberculosis represents a potential target for antituberculosis drugs. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Previously, we determined the crystal structure of PS from M. tuberculosis and its complexes with AMPCPP, pantoate, and pantoyl adenylate. Here, we describe the crystal structure of this enzyme complexed with AMP and its last substrate, beta-alanine, and show that the phosphate group of AMP serves as an anchor for the binding of beta-alanine. This structure confirms that binding of beta-alanine in the active site cavity can occur only after formation of the pantoyl adenylate intermediate. A new crystal form was also obtained; it displays the flexible wall of the active site cavity in a conformation incapable of binding pantoate. Soaking of this crystal form with ATP and pantoate gives a fully occupied complex of PS with ATP. Crystal structures of these complexes with substrates, the reaction intermediate, and the reaction product AMP provide a step-by-step view of the PS-catalyzed reaction. A detailed reaction mechanism and its implications for inhibitor design are discussed.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an alpha-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product alpha-ketobutyrate, and two d-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 A) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the alpha-proton from d-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the l-amino acid substrates and may account for the stereospecificity of this enzyme toward d-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S beta-methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis.

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395(T) in Escherichia coli. The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5'-phosphate. On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to L-serine and product, and showed the following new aspects: First, we analyzed the binding of L-serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex. Second, various serine analogues were also examined; the data indicated that the alpha-carboxyl group of L-serine was quite important for substrate recognition by SPT. Third, we focused on a series of SPT inhibitors, which have been used as convenient tools to study the cell responses caused by sphingolipid depletion. The interaction of SPT with myriocin suggested that such product-related compounds would strongly and competitively inhibit enzyme activity by forming an external aldimine in the active site of the enzyme. Beta-chloro-L-alanine and L-cycloserine were found to generate characteristic PLP-adducts that produced inactivation of SPT in an irreversible manner. The detailed mechanisms for the SPT inactivation were discussed. This is the first analysis of the inhibition mechanisms of SPT by these compounds, which will provide an enzymological basis for the interpretation of the results from cell biological experiments.

The C-terminal thioesterase domain of the nonribosomal peptide synthetase producing the lipopetide surfactin (Srf TE) retains autonomous ability to generate the cyclic peptidolactone skeleton of surfactin when provided with a soluble beta-hydroxy-butyryl-heptapeptidyl thioester substrate. Utilizing the recently solved crystal structure [Bruner, S. D., et al. (2002) Structure 10, 301-310], the active-site nucleophile, Ser80, was changed to Cys, and the other members of the catalytic triad, Asp107 and His207, were changed to Ala, with the resulting mutants lacking detectable activity. Two cationic side chains in the active site, Lys111 and Arg120, were changed to Ala, causing an increased partitioning of the product to hydrolysis, as did a P26G mutant, mimicking the behavior of lipases. To evaluate recognition elements in substrates used by Srf TE, alterations to the fatty acyl group, the heptapeptide, and the thioester leaving group were made, and the resulting substrates were characterized for kinetic competency and flux of product to cyclization or hydrolysis. Alterations that could be accepted for cyclization were identified in all three parts of the substrate, although tolerance limits for changes varied. In addition, cocrystal structures of Srf TE with dipeptidyl boronate inhibitors were solved, illustrating the critical binding determinants of the substrate. On the basis of the structures and biochemical data, the cyclizing conformation of the surfactin peptide was modeled into the enzyme active site.

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.

The oxidation of Co(II) bleomycin A2 by dioxygen leads to two products, HO2-Co(III) bleomycin A2 (form I) and Co(III) bleomycin A2 (form II). 1H NMR chemical shift assignments for protons of both forms have been made by two-dimensional NMR spectral techniques. The chemical shifts of protons throughout forms I and II differ from each other and from apobleomycin A2. NOESY spectra reveal a number of intermediate and long-range 1H-1H couplings within the metal-binding domain, between the metal-binding domain and the peptide linker, which connects it and the DNA-binding region of the molecule, and, in form I, between the DNA- and metal-binding domains. Molecular dynamics calculations were carried out based on the NOESY results and an adjustable square pyramidyl ligand geometry around Co(III) composed of nitrogen atoms of the primary and secondary amine groups, pyridine (N5), and amide and imidazole (N1) of the hydroxyhistidine residue. In form I, the bithiazole group folds back across the square pyramid forming a compact structure. Although this conformational feature was not observed in form II, the peptide linker between the metal- and DNA-binding domains in both species shows extensive folding based on a large number of intramolecular interactions.

The carbodiimide-mediated reaction of N-hydroxysuccinimide with carboxyl groups immobilized to hydroxyl-containing polymers (such as Sepharose or Trisacryl) leads to an undesirable side reaction in high yields. The product of this reaction interferes with the application of such columns for further affinity-based purification. In addition to the desired N-hydroxysuccinimide ester, a bis(N-hydroxysuccinimide)derivative of beta-alanine [namely, N- [(succinimidooxy)carbonyl]-beta-alanine N-hydroxysuccinimide ester] is probably produced that reacts subsequently with the hydroxyl group of the polymer via ester and carbamate bonds. These beta-alanine derivatives are formed upon interaction of dicyclohexylcarbodiimide with 3 equiv of N-hydroxysuccinimide followed by a Lossen rearrangement. The amount of beta-alanine thus coupled is very high compared to the number of carboxyl groups present on the resin. The beta-alanine bound through the ester bond comprises about 90% of the beta-alanine bound. Alkaline treatment of the ester-bonded beta-alanine-containing polymers (prior to coupling of amino-containing ligands) causes a rearrangement yielding beta-alanine with a free carboxyl group coupled through a stable carbamate linkage. After coupling of amino-containing ligands, the above-described rearrangement cannot occur, and the beta-alanine-linked ligand leaks from the polymer via hydrolysis of the ester bond. The newly formed carboxyl groups (derived from the rearrangement) can be used to prepare active esters. In view of the above, we developed methods for the preparation of nitrophenyl esters as well as N-hydroxysuccinimide esters free of unstable beta-alanine derivatives on polymers containing hydroxyl groups. Upon coupling with amino-containing ligands, these esters yield resins bearing chemically stable bonds.