Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4′-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4′-hydroxylation and further decreased by 4′-methoxylation against mushroom tyrosinase. Surprisingly, 4′-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4′-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4′-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors.     

Indirect oxidation of amino acid phenylhydrazides by mushroom tyrosinase

We have investigated oxidation of amino acid phenylhydrazides by mushroom tyrosinase in the presence of 4-tert-butylcatechol and N-acetyl-L-tyrosine. Spectrophotometric measurements showed gradual disappearance of 4-tert-butyl-o-benzoquinone, generated by oxidation of 4-tert-butylcatechol with sodium periodate, after addition of amino acid phenylhydrazides. However, the presence of the phenylhydrazides did not influence the concentration of 4-tert-butyl-o-benzoquinone formed during enzymatic oxidation. Oxygen consumption measurements demonstrated that in a mixture both compounds were oxidized but the reaction rate was proportional to the concentration of the catechol. In the oxidation of N-acetyl-L-tyrosine addition of phenylhydrazides shortened the lag period, indicating that they acted as reducing agents, converting N-acetyl-L-dopaquinone to N-acetyl-L-dopa. In HPLC analysis of the oxidation 4-tert-butylcatechol and the phenylhydrazide of Boc-tryptophan only the N-protected amino acid and 4-tert-butyl-o-benzoquinone were detected as final products. In the presence of the natural substrates the oxidation of amino acid phenylhydrazides required much smaller amounts of the enzyme and was up to 40 times faster than the reaction carried out without these compounds. These results demonstrate that tyrosinase can oxidize phenylhydrazides indirectly through o-quinones. This reaction explains the inhibitory effect of agaritine, a natural amino acid hydrazide, on melanin formation and the inhibitory effects of other hydrazine derivatives on tyrosinase described in the literature.     

Structural insights into the regulation of aromatic amino acid hydroxylation

1. Download : Download high-res image (533KB)
2. Download : Download full-size image

     

Accurate prediction of hot spot residues through physicochemical characteristics of amino acid sequences

Hot spot residues of proteins are fundamental interface residues that help proteins perform their functions. Detecting hot spots by experimental methods is costly and time‐consuming. Sequential and structural information has been widely used in the computational prediction of hot spots. However, structural information is not always available. In this article, we investigated the problem of identifying hot spots using only physicochemical characteristics extracted from amino acid sequences. We first extracted 132 relatively independent physicochemical features from a set of the 544 properties in AAindex1, an amino acid index database. Each feature was utilized to train a classification model with a novel encoding schema for hot spot prediction by the IBk algorithm, an extension of the K‐nearest neighbor algorithm. The combinations of the individual classifiers were explored and the classifiers that appeared frequently in the top performing combinations were selected. The hot spot predictor was built based on an ensemble of these classifiers and to work in a voting manner. Experimental results demonstrated that our method effectively exploited the feature space and allowed flexible weights of features for different queries. On the commonly used hot spot benchmark sets, our method significantly outperformed other machine learning algorithms and state‐of‐the‐art hot spot predictors. The program is available at http://sfb.kaust.edu.sa/pages/software.aspx . Proteins 2013; 81:1351–1362 © 2013 Wiley Periodicals, Inc.     

Structural insights into sialic acid enzymology

Sialic acids are a diverse family of negatively charged sugars that play essential biological roles. Their presence and relative abundance in different cells is ultimately regulated by the concerted action of a large set of enzymes. In this review, we focus on the most recent advances on the enzymes that govern sialic acid metabolism, with emphasis on structural work. Major progress has been made in dissecting the catalytic mechanism of sialidases, revealing a modified scenario of the typical glycosidase ping-pong mechanism. Similarly, X-ray structures of sialyltransferases uncover significant variations of formerly known glycosyltransferase foldings. Both sialidases and sialyltransferases seem to tell us that sialic acid-handling enzymes have evolved important modifications related to the distinctive features of sialic acid itself.     

Structural and mechanistic insights into the oxy form of tyrosinase from molecular dynamics simulations

The first, long time scale (16-ns) ligand field molecular dynamics (LFMD) simulations of the oxy form of tyrosinase are reported. The calculations use our existing type 3 copper force field for the peroxido-bridged [Cu₂O₂]²⁺ unit which is here translated from MMFF into the AMBER format together with a new charge scheme. The protein secondary and tertiary structures are not significantly altered by removing the ‘caddie’ protein, ORF378, which must be bound to tyrosinase before crystals will grow. A comprehensive principal component analysis of the Cartesian coordinates from the final 8 ns shows that the protein backbone is relatively rigid. However, the significant butterfly fold of the [Cu₂O₂]²⁺ moiety observed in the X-ray structure, presumably due to the caddie protein tyrosine at the active site, is absent in the simulations. LFMD gives a clear and persistent distinction between equatorial and axial Cu–N distances, with the latter about 0.2 Å longer and remaining syn to each other. However, the two coordination spheres display important differences. LFMD simulations of the symmetric model complex [μ-η²:μ²-O₂{Cu(Meim)₃}₂]²⁺ (Meim is 5-methyl-1H-imidazole) provide a mechanism for syn–anti interchange of axial ligands which suggests, in combination with the old experimental X-ray data, the new LFMD simulations and traditional coordination chemistry arguments, that His₅₄ on Cu_A is ‘insipiently axial’ and that a combination of a butterfly distortion of the [Cu₂O₂]²⁺ group and a rotation of the Cu_A(His)₃ moiety converts the vacant, initially axial, binding site on Cu_A into a much more favourable equatorial site.     

Identification of hot spot residues at protein-protein interface

It is known that binding free energy of protein-protein interaction is mainly contributed by hot spot (high energy) interface residues. Here, we investigate the characteristics of hot spots by examining inter-atomic sidechain-sidechain interactions using a dataset of 296 alanine-mutated interface residues. Results show that hot spots participate in strong and energetically favorable sidechain-sidechain interactions. Subsequently, we describe a novel, yet simple 'hot spot' prediction model with an accuracy that is similar to many available approaches. The model is also shown to efficiently distinguish specific protein-protein interactions from non-specific interactions.     

Structural insights into the role of the ACTH receptor cysteine residues on receptor function

The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH(2) terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.     

New insights into the evolution of metazoan tyrosinase gene family

Tyrosinases, widely distributed among animals, plants and fungi, are involved in the biosynthesis of melanin, a pigment that has been exploited, in the course of evolution, to serve different functions. We conducted a deep evolutionary analysis of tyrosinase family amongst metazoa, thanks to the availability of new sequenced genomes, assessing that tyrosinases (tyr) represent a distinctive feature of all the organisms included in our study and, interestingly, they show an independent expansion in most of the analyzed phyla. Tyrosinase-related proteins (tyrp), which derive from tyr but show distinct key residues in the catalytic domain, constitute an invention of chordate lineage. In addition we here reported a detailed study of the expression territories of the ascidian Ciona intestinalis tyr and tyrps. Furthermore, we put efforts in the identification of the regulatory sequences responsible for their expression in pigment cell lineage. Collectively, the results reported here enlarge our knowledge about the tyrosinase gene family as valuable resource for understanding the genetic components involved in pigment cells evolution and development.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Resonant recognition model of neuropeptide Y family: hot spot amino acid distribution in the sequences

The resonant recognition model is used to predict structurally and functionally important amino acid residues (so-called hot spots) in the neuropeptide Y (NPY) family. Thirty-three polypeptides belong to this family. All of them consist of 36 amino acids. The model predicts that residues 10 and 28 in the polypeptides are hot spots. In the 33 polypeptides, most of the amino acids at residue 10 are acidic amino acids, glutamic acid and aspartic acid. Other minor amino acids, serine, glycine, and proline, have high probabilities of -turn occurrence. Amino acids at residue 28 are all branched hydrophobic amino acids, isoleucine, leucine, and valine. The profile for predicting hot spots indicates repeating patterns of residues 1–18 and residues 19–36. Absolute values at residue i and residue i + 18 are the same, but these residues have opposite signs. Therefore the model of the NPY family predicts hot spots concerning a combination of residue i and residue i + 18.     

Structural insights into fusidic acid resistance and sensitivity in EF-G

Fusidic acid (FA) is a steroid antibiotic commonly used against Gram positive bacterial infections. It inhibits protein synthesis by stalling elongation factor G (EF-G) on the ribosome after translocation. A significant number of the mutations conferring strong FA resistance have been mapped at the interfaces between domains G, III and V of EF-G. However, direct information on how such mutations affect the structure has hitherto not been available. Here we present the crystal structures of two mutants of Thermus thermophilus EF-G, G16V and T84A, which exhibit FA hypersensitivity and resistance in vitro, respectively. These mutants also have higher and lower affinity for GTP respectively than wild-type EF-G. The mutations cause significant conformational changes in the switch II loop that have opposite effects on the position of a key residue, Phe90, which undergoes large conformational changes. This correlates with the importance of Phe90 in FA sensitivity reported in previous studies. These structures substantiate the importance of the domain G/domain III/domain V interfaces as a key component of the FA binding site. The mutations also cause subtle changes in the environment of the "P-loop lysine", Lys25. This led us to examine the conformation of the equivalent residue in all structures of translational GTPases, which revealed that EF-G and eEF2 form a group separate from the others and suggested that the role of Lys25 may be different in the two groups.     

Theoretical insights into the properties of amino acid ionic liquids in aqueous solution

This report presents a systematic investigation of the interactions of water molecule(s) with a series of amino acid cations (Gly⁺, Ala⁺, Val⁺, and Leu⁺), halogen anions (Cl^?, Br^?, BF₄ ^?, and PF₆ ^?), and clusters (GlyCl) _n (n?=?1–5). The results reveal that H-bonds between amino acid ionic liquids (AAILs) and water molecules are crucial to the properties of aqueous solution of AAILs. The properties of AAIL in water solution depend on the alkyl chain of the amino acid cation, the size of the halogen anion, and the number of water molecules, which provides a certain theoretical basis for the design and application of new AAILs. A series of calculations for some different models showed that quadruple-GlyCl hydrate represents a basic unit for the Gly–water binary system, and can be employed as the simplest model for studying an AAIL–water cluster. On the basis of this model, the effects of water on the hygroscopicity, speed of solubility, viscosity, density, solution enthalpy, and polarity of the AAIL were also predicted. Most importantly, unlike traditional ILs, the novel GlyCl-type AAIL favors interaction of its cationic part, rather than its anionic part, with surrounding water molecules, thus amino acid cationic ILs expand the types of IL available, increasing the choice of ILs for different purposes. We hope that the application of this AAIL in many fields will lead to optimization of this class of compound and be of benefit to the environment.

Open image in new window

Graphical Abstract Quadruple-GlyCl hydrate represents the basic unit for a GlyCl-water binary system, which can be employed as the simplest model for studying an amino acid ionic liquid (AAIL)-water cluster. The effects of available water on some properties of AAIL are predicted. GlyCl-type AAIL is a novel IL, which prefers its cationic part over its anionic part for interaction with surrounding water molecules. The properties of AAIL in water solution can be adjusted by varying the ion used and the solvent.

     

Structural insights into the clathrin coat

Clathrin is a cytoplasmic protein best known for its role in endocytosis and intracellular trafficking. The diverse nature of clathrin has recently become apparent, with strong evidence available suggesting roles in both chromosome segregation and reassembly of the Golgi apparatus during mitosis. Clathrin functions as a heterohexamer, adopting a three-legged triskelion structure of three clathrin light chains and three heavy chains. During endocytosis clathrin forms a supportive network about the invaginating membrane, interacting with itself and numerous adapter proteins. Advances in the field of structural biology have led us to a greater understanding of clathrin in its assembled state, the clathrin lattice. Combining techniques such as X-ray crystallography, NMR, and cryo-electron microscopy has allowed us to piece together the intricate nature of clathrin-coated vesicles and the interactions of clathrin with its many binding partners. In this review I outline the roles of clathrin within the cell and the recent structural advances that have improved our understanding of clathrin-clathrin and clathrin-protein interactions.     

New insights into the regulation of plant immunity by amino acid metabolic pathways

Besides defence pathways regulated by classical stress hormones, distinct amino acid metabolic pathways constitute integral parts of the plant immune system. Mutations in several genes involved in Asp‐derived amino acid biosynthetic pathways can have profound impact on plant resistance to specific pathogen types. For instance, amino acid imbalances associated with homoserine or threonine accumulation elevate plant immunity to oomycete pathogens but not to pathogenic fungi or bacteria. The catabolism of Lys produces the immune signal pipecolic acid (Pip), a cyclic, non‐protein amino acid. Pip amplifies plant defence responses and acts as a critical regulator of plant systemic acquired resistance, defence priming and local resistance to bacterial pathogens. Asp‐derived pyridine nucleotides influence both pre‐ and post‐invasion immunity, and the catabolism of branched chain amino acids appears to affect plant resistance to distinct pathogen classes by modulating crosstalk of salicylic acid‐ and jasmonic acid‐regulated defence pathways. It also emerges that, besides polyamine oxidation and NADPH oxidase, Pro metabolism is involved in the oxidative burst and the hypersensitive response associated with avirulent pathogen recognition. Moreover, the acylation of amino acids can control plant resistance to pathogens and pests by the formation of protective plant metabolites or by the modulation of plant hormone activity.     

Structural insights into the SNARE mechanism

Eukaryotic cells distribute materials among intracellular organelles and secrete into the extracellular space through cargo-loaded vesicles. A concluding step during vesicular transport is the fusion of a transport vesicle with a target membrane. SNARE proteins are essential for all vesicular fusion steps, thus they possibly comprise a conserved membrane fusion machinery. According to the "zipper" model, they assemble into stable membrane-bridging complexes that gradually bring membranes in juxtaposition. Hence, complex formation may provide the necessary energy for overcoming the repulsive forces between two membranes. During the last years, detailed structural and functional studies have extended the evidence that SNAREs are mostly in accord with the zipper model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.     

Peptide and amino acid glycation: new insights into the Maillard reaction.

Nonenzymatic glycation of proteins, peptides and other macromolecules (the Maillard reaction) has been implicated in a number of pathologies, most clearly in diabetes mellitus. but also in the normal processes of aging and neurodegenerative amyloid diseases such as Alzheimer's. In the early stage, glycation results in the formation of Amadori-modified proteins. In the later stages, advanced glycation end products (AGE) are irreversibly formed from Amadori products leading to the formation of reactive intermediates, crosslinking of proteins, and the formation of brown and fluorescent polymeric materials. Although, the glycation of structural proteins has been attributed a key role in the complications of diabetes, recent attention has been devoted to the physiological significance of glycated peptide hormones. This review focuses on the physico-chemical properties of the Amadori compounds of bioactive peptides of endogenous and exogenous origin, such as Leu-enkephalin and morphiceptin, investigated under different conditions as well as on novel pathways in the Maillard reaction observed from investigating intramolecular events in ester-linked glycopeptides.