In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum

The purple chromobacterial pigment violacein arises by enzymatic oxidation and coupling of two molecules of l-tryptophan to give a rearranged pyrrolidone-containing scaffold in the final pigment. We have purified five contiguously encoded proteins VioA-E after expression in Escherichia coli and demonstrate the full 14-electron oxidation pathway to yield the final chromophore. The flavoenzyme VioA and the heme protein VioB work in conjunction to oxidize and dimerize l-tryptophan to a nascent product that can default to the off pathway metabolite chromopyrrolic acid. In the presence of VioE, the intermediate instead undergoes on-pathway [1,2] indole rearrangement to prodeoxyviolacein. The last two enzymes in the pathway are flavin-dependent oxygenases, VioC and VioD, that act sequentially. VioD hydroxylates one indole ring at the 5-position to yield proviolacein, and VioC then acts on the other indole ring at the 2-position to create the oxindole and complete violacein formation.     

The violacein biosynthetic enzyme VioE shares a fold with lipoprotein transporter proteins

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. Our 1.21 A resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB. For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE have identified the active site and five residues important for binding or catalysis. This structural and mutagenesis study suggests that VioE acts as a catalytic chaperone, using a fold previously associated with lipoprotein transporters to catalyze the production of its prodeoxyviolacein product.     

Molecular docking study of the interactions between the thioesterase domain of human fatty acid synthase and its ligands

Human fatty acid synthase (hFAS) thioesterase domain (TE) is an attractive drug target to treat obesity and cancer. On the basis of the recently published crystal structure of TE domain of hFAS, we performed molecular surface analysis and docking study to characterize the molecular interactions between the enzyme and its various ligands. Surface analysis identified the ligand-binding pocket of TE domain that encompasses the catalytic triad of Ser2308, His2481, Asp2338. Docking of palmitate, the main biological product of hFAS, into this pocket revealed the ligand-binding mode, in which the hydrophobic interactions are the dominant driving forces. The catalytic mechanism of TE domain can also be well explained based on the generated TE-palmitate complex structure. Moreover, the comparison of the binding modes of five fatty acids with chain lengths ranging from 12 to 20 carbons confirmed that the ligand binding pocket of TE domain is a decisive factor in chain length specificity. In addition, docking of two known TE inhibitors, c75 and orlistat revealed the pharmacophore of these hFAS TE inhibitors, which will prove useful in structure-based drug design against this important target.     

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme-product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole-time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins.     

Molecular dynamics simulations of the resting and hydrogen peroxide-bound states of cytochrome c peroxidase.

The current hypothesis for the formation of the catalytically active compound I of peroxidases from the resting state and peroxide involves formation of a reversible "inner-sphere" complex in which the peroxide is bound to the heme iron. It is this precursor that is postulated to then form compound I. However, this crucial putative transient intermediate has not yet been definitively detected or characterized by experimental methods. We report here the use of energy minimization and molecular dynamics simulation together with the known X-ray structure of cytochrome c peroxidase to investigate the nature of this complex and comparisons of it with the resting state in which a water is bound as a ligand. Among the properties monitored in these simulations are the mode of binding of the peroxide to the heme iron, its interactions with neighboring amino acid residues, and the extent to which the binding of the peroxide perturbs both the local environment around the heme unit and more distant regions. The results of this study indicate that solvated, full protein dynamics is required to obtain reliable results for the known resting-state complex and hence for the uncharacterized peroxide complex. In this complex, the peroxide binds to the heme iron in a dynamically averaged end-on fashion, rather than a bridged structure, with approximately equal probability of each oxygen serving as the ligand to the iron. Binding of the peroxide as a ligand disrupts the H-bonded network of waters in the distal binding pocket which are present in the resting state, but there is no dramatic perturbation of the nearby amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural insights into ligand-binding pocket formation in Nurr1 by molecular dynamics simulations

Abstract

The nuclear receptor Nurr1 (NR4A2) has been identified as a potential target for the treatment of Parkinson’s disease. In contrast to most other nuclear receptors, the X-ray crystal structure of the Nurr1 ligand-binding domain (LBD) lacks any ligand-binding pocket (LBP). However, NMR spectroscopy measurements have revealed that the known Nurr1 agonist docosahexaenoic acid (DHA) binds to a region within the LBD that corresponds to the classical NR ligand-binding pocket (LBP). In order to investigate the structural dynamics of the Nurr1 LBD and to study potential LBP formation, the conformational space of the receptor was sampled using a molecular dynamics (MD) simulation. Docking of DHA into 50,000 LBD structures extracted from the simulation revealed the existence of a transient LBP that is capable to fully harbor the compound. The location of the identified pocket overlaps with the ligand-binding site suggested by NMR experiments. Structural analysis of the protein-ligand complex showed that only modest structural rearrangements within the Nurr1 LBD are required for LBP formation. These findings may support structure-based drug discovery campaigns for the development of receptor-specific agonists.     

Biosynthesis of a trypanocide by Chromobacterium violaceum

Radio-isotope studies indicated not only that l-tryptophan can serve as carbon source for synthesis of the trypanocide, violacein by Chromobacterium violaceum (BB-78 strain) but also that isatin and indole 3-acetic acid are both important metabolic intermediates. Using 3-indolyl [2-¹⁴C] and [1-¹⁴C] acetic acid, it was found that the carboxylic carbon was not eliminated and that indole-3-acetic acid was incorporated intact into the pigment structure. N-Ethyl(5-hydroxy-indol-3-yl)-2-indolylethylamide is also an important metabolic intermediate in the violacein biosynthesis. This is the first report of a metabolic scheme for violacein synthesis which includes an intermediate other than l-tryptophan.     

Partially glycosylated dendrimers block MD-2 and prevent TLR4-MD-2-LPS complex mediated cytokine responses

The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.     

Violacein and related tryptophan metabolites produced by <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis>: biosynthetic mechanism and pathway for construction of violacein core

Violacein is a natural violet pigment produced by several Gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, and Pseudoalteromonas tunicata D2, among others. This pigment has potential medical applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. The structure of violacein consists of three units: a 5-hydroxyindole, an oxindole, and a 2-pyrrolidone. The biosynthetic origins of hydrogen, nitrogen, and carbon in the pyrrolidone nucleus were established by feeding experiments using various stable isotopically labeled tryptophans (Trps). Pro-S hydrogen of CH₂ at the 3-position of Trp is retained during biosynthesis. The nitrogen atom is exclusively from the α-amino group, and the skeletal carbon atoms originate from the side chains of the two Trp molecules. All three oxygen atoms in the violacein core are derived from molecular oxygen. The most interesting biosynthetic mechanism is the 1,2-shift of the indole nucleus on the left side of the violacein scaffold. The alternative Trp molecule is directly incorporated into the right side of the violacein core. This indole shift has been observed only in violacein biosynthesis, despite the large number of natural products having been isolated. There were remarkable advances in biosynthetic studies in 2006–2008. During the 3 years, most of the intermediates and the complete pathway were established. Two independent processes are involved: the enzymatic process catalyzed by the five proteins VioABCDE or the alternative nonenzymatic oxidative decarboxylation reactions. The X-ray crystallographic structure of VioE that mediates the indole rearrangement reaction was recently identified, and the mechanism of the indole shift is discussed here.     

Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme

The 5' capping of mammalian pre-mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 A crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5' triphosphate end. Structural, biochemical and mutational results show that despite sharing an HCxxxxxR(S/T) motif, a phosphoenzyme intermediate and a core alpha/beta-fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by mammalian capping enzyme differs from that used by protein phosphatases to hydrolyze phosphomonoesters. The most significant difference is the absence of a carboxylate general acid catalyst in RNA triphosphatase. Residues conserved uniquely among the RNA phosphatase subfamily are important for function in cap formation and are likely to play a role in substrate recognition.     

Reaction of porcine pancreatic elastase with 7-substituted 3-alkoxy-4-chloroisocoumarins: design of potent inhibitors using the crystal structure of the complex formed with 4-chloro-3-ethoxy-7-guanidinoisocoumarin

The crystal structure of the acyl enzyme formed upon inhibition of porcine pancreatic elastase (PPE) by 4-chloro-3-ethoxy-7-guanidinoisocoumarin has been determined at a 1.85-A effective resolution. The chlorine atom is still present in this acyl enzyme, in contrast to the previously reported structure of the 7-amino-4-chloro-3-methoxyisocoumarin-PPE complex where the chlorine atom has been replaced by an acetoxy group. The guanidino group forms hydrogen bonds with the carbonyl group and side-chain hydroxyl group of Thr-41, and the acyl carbonyl group has been twisted out of the oxyanion hole. Molecular modeling indicates that the orientation of the initial Michaelis enzyme-inhibitor complex is quite different from that of the acyl enzyme since simple reconstruction of the isocoumarin ring would result in unfavorable interactions with Ser-195 and His-57. Molecular models were used to design a series of new 7-(alkylureido)- and 7-(alkylthioureido)-substituted derivatives of 3-alkoxy-7-amino-4-chloroisocoumarin as PPE inhibitors. All the 3-ethoxyisocoumarins were better inhibitors than those in the 3-methoxy series due to better interactions with the S1 pocket of PPE. The best ureido inhibitor also contained a tert-butylureido group at the 7-position of the isocoumarin. Due to a predicted interaction with a small hydrophobic pocket on the surface of PPE, this isocoumarin and a related phenylthioureido derivative are among the best irreversible inhibitors thus far reported for PPE (kobs/[I] = 8100 M-1 s-1 and 12,000 M-1 s-1). Kinetic studies of the stability of enzyme-inhibitor complexes suggest that many isocoumarins are alkylating the active site histidine at pH 7.5 via a quinone imine methide intermediate, while at pH 5.0, the predominant pathway appears to be simple formation of a stable acyl enzyme derivative.     

DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family: ASP171 replaces the distal histidine of classical peroxidases

DyP, a unique dye-decolorizing enzyme from the fungus Thanatephorus cucumeris Dec 1, has been classified as a peroxidase but lacks homology to almost all other known plant peroxidases. The primary structure of DyP shows moderate sequence homology to only two known proteins: the peroxide-dependent phenol oxidase, TAP, and the hypothetical peroxidase, cpop21. Here, we show the first crystal structure of DyP and reveal that this protein has a unique tertiary structure with a distal heme region that differs from that of most other peroxidases. DyP lacks an important histidine residue known to assist in the formation of a Fe4+ oxoferryl center and a porphyrin-based cation radical intermediate (compound I) during the action of ubiquitous peroxidases. Instead, our tertiary structural and spectrophotometric analyses of DyP suggest that an aspartic acid and an arginine are involved in the formation of compound I. Sequence analysis reveals that the important aspartic acid and arginine mentioned above and histidine of the heme ligand are conserved among DyP, TAP, and cpop21, and structural and phylogenetic analyses confirmed that these three enzymes do not belong to any other families of peroxidase. These findings, which strongly suggest that DyP is a representative heme peroxidase from a novel family, should facilitate the identification of additional new family members and accelerate the classification of this novel peroxidase family.     

Structural basis for the inhibition of Aurora A kinase by a novel class of high affinity disubstituted pyrimidine inhibitors

The 2.25 A crystal structure of a complex of Aurora A kinase (AIKA) with cyclopropanecarboxylic acid-(3-(4-(3-trifluoromethyl-phenylamino)-pyrimidin-2-ylamino)-phenyl)-amide 1 is described here. The inhibitor binding mode is novel, with the cyclopropanecarboxylic acid moiety directed towards the solvent exposed region of the ATP-binding pocket, and several induced structural changes in the active-site compared with other published AIK structures. This structure provides context for the available SAR data on this compound class, and could be exploited for the design of analogs with increased affinity and selectivity for AIK.     

Dimerization Interface of 3-Hydroxyacyl-CoA Dehydrogenase Tunes the Formation of Its Catalytic Intermediate

3-hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.35) is a homodimeric enzyme localized in the mitochondrial matrix, which catalyzes the third step in fatty acid β-oxidation. The crystal structures of human HAD and subsequent complexes with cofactor/substrate enabled better understanding of HAD catalytic mechanism. However, numerous human diseases were found related to mutations at HAD dimerization interface that is away from the catalytic pocket. The role of HAD dimerization in its catalytic activity needs to be elucidated. Here, we solved the crystal structure of Caenorhabditis elegans HAD (cHAD) that is highly conserved to human HAD. Even though the cHAD mutants (R204A, Y209A and R204A/Y209A) with attenuated interactions on the dimerization interface still maintain a dimerization form, their enzymatic activities significantly decrease compared to that of the wild type. Such reduced activities are in consistency with the reduced ratios of the catalytic intermediate formation. Further molecular dynamics simulations results reveal that the alteration of the dimerization interface will increase the fluctuation of a distal region (a.a. 60–80) that plays an important role in the substrate binding. The increased fluctuation decreases the stability of the catalytic intermediate formation, and therefore the enzymatic activity is attenuated. Our study reveals the molecular mechanism about the essential role of the HAD dimerization interface in its catalytic activity via allosteric effects.     

Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor

The pregnane X receptor (PXR) detects the presence of a wide variety of endogenous and xenobiotic compounds, and is a master regulator of the expression of genes central to drug metabolism and excretion. We present the 2.0A crystal structure of the human PXR ligand-binding domain (LBD) in complex with the cholesterol-lowering compound SR12813 and a 25 amino acid residue fragment of the human steroid receptor coactivator-1 (SRC-1) containing one LXXLL motif. PXR crystallizes as a homodimer in the asymmetric unit in this structure and possesses a novel alpha2 helix adjacent to its ligand-binding cavity. The SRC-1 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 helix on the surface of PXR. In contrast with previous PXR structures, in which SR12813 bound in multiple orientations, the small SR12813 agonist in this structure binds in a single, unique orientation within the receptor's ligand-binding pocket and contacts the AF-2 helix. Thermal denaturation studies reveal that the SR12813 ligand and SRC-1 coactivator peptide each stabilize the LBD of PXR, and that together they exert an additive effect on the stability of the receptor. These results indicate that the binding of coactivator to the surface of PXR limits the ability of this promiscuous receptor to "breathe" and helps to trap a single, active conformation of SR12813. They further reveal that specificity is required for PXR activation.     

Structure of an anti-Lewis X Fab fragment in complex with its Lewis X antigen

The Lewis X trisaccharide is pivotal in mediating specific cell-cell interactions. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, has been shown to recognize the Lewis X trisaccharide. Here we describe the structure of the Fab fragment of 291-2G3-A, with Lewis X, to 1.8 A resolution. The crystallographic analysis revealed that the antigen binding site is a rather shallow binding pocket, and residues from all six complementary determining regions of the antibody contact all sugar residues. The high specificity of the binding pocket does not result in high affinity; the K(D) determined by isothermal calorimetry is 11 microM. However, this affinity is in the same range as for other sugar-antibody complexes. The detailed understanding of the antibody-Lewis X interaction revealed by the crystal structure may be helpful in the design of better diagnostic tools for schistosomiasis and for studying Lewis X-mediated cell-cell interactions by antibody interference.     

Observation of an arsenic adduct in an acetyl esterase crystal structure

The crystal structures of an acetyl esterase, HerE, and its complex with an inhibitor dimethylarsinic acid have been determined at 1.30- and 1.45-A resolution, respectively. Although the natural substrate for the enzyme is unknown, HerE hydrolyzes the acetyl groups from heroin to yield morphine and from phenyl acetate to yield phenol. Recently, the activity of the enzyme toward heroin has been exploited to develop a heroin biosensor, which affords higher sensitivity than other currently available detection methods. The crystal structure reveals a single domain with the canonical alpha/beta hydrolase fold with an acyl binding pocket that snugly accommodates the acetyl substituent of the substrate and three backbone amides that form a tripartite oxyanion hole. In addition, a covalent adduct was observed between the active site serine and dimethylarsinic acid, which inhibits the enzyme. This crystal structure provides the first example of an As-containing compound in a serine esterase active site and the first example of covalent modification of serine by arsenic. Thus, the HerE complex reveals the structural basis for the broad scope inhibition of serine hydrolases by As(V)-containing organic compounds.     

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.