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Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtualscreening (VS) protocol by experimentally testing the ''in vitro'' activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with Ki values ⩾10 μM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with Ki values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a Ki value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery. [ABSTRACT FROM AUTHOR]

Background: In recent years, an exponential growing number of tools for protein sequence analysis, editing and modeling tasks have been put at the disposal of the scientific community. Despite the vast majority of these tools have been released as open source software, their deep learning curves often discourages even the most experienced users. Results: A simple and intuitive interface, PyMod, between the popular molecular graphics system PyMOL and several other tools (i.e., [PSI-]BLAST, ClustalW, MUSCLE, CEalign and MODELLER) has been developed, to show how the integration of the individual steps required for homology modeling and sequence/structure analysis within the PyMOL framework can hugely simplify these tasks. Sequence similarity searches, multiple sequence and structural alignments generation and editing, and even the possibility to merge sequence and structure alignments have been implemented in PyMod, with the aim of creating a simple, yet powerful tool for sequence and structure analysis and building of homology models. Conclusions: PyMod represents a new tool for the analysis and the manipulation of protein sequences and structures. The ease of use, integration with many sequence retrieving and alignment tools and PyMOL, one of the most used molecular visualization system, are the key features of this tool. Source code, installation instructions, video tutorials and a user's guide are freely available at the URL http://schubert.bio.uniroma1.it/pymod/index.html [ABSTRACT FROM AUTHOR]

Background: A wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures. Results: The construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions. Conclusion: This study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in the protein core and/or a better packing of alpha-helices with the rest of the structure, caused by the presence of hydrophobic beta-branched side chains. [ABSTRACT FROM AUTHOR]

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly ( r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the coenzyme-binding site of serine transhydroxymethylase from rabbit liver has been determined. After reduction with NaBH4 and aminoethylation, a first sample of enzyme was digested with thermolysin and a single phosphopyridoxyl peptide was isolated. A second sample of similarly treated enzyme was digested with chymotrypsin and three phosphopyridoxyl peptides clearly originating from a unique coenzyme-binding site were isolated. Sequence analysis of these peptides indicate the following structure: Val-Val-Thr-Thr-Thr-His-Lys(Pxy)-Thr-Leu. Sequence homologies of the active site of various pyridoxalphosphate enzymes are discussed in terms of a possible catalytic role and of evolution of this class of proteins. [ABSTRACT FROM AUTHOR]

A method has been developed which allows isolation of 0,3–0,5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts: the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, soIutions and from solutions of ammonium sulphate. The amino acid composition. N and C-terminal amino acid sequences and subunit molecular weight have been determined: these characteristic properties are compared with those of the cytoplasmic isozyme from the same source. [ABSTRACT FROM AUTHOR]

1. A primary structure is presented for mitochondrial aspartate aminotransferase from pig heart based on previously published results [Barra et al. (1977) FEBS Lett. 83, 241-244; Doonan et al. (1979) Ital. J. Biochem. 28, 441-455; Barra et al. (1979) Ital. J. Biochem. 28, 456-477, 478-490] and a small amount of extra information given here. 2. A detailed comparison is made between the primary structures of the cytosolic and mitochondrial isozymes. These are homologous proteins with 47% identity of primary structure and do not differ appreciably in overall polarity. 3. Hydrophobic chromatography of the isozymes using alkyl agaroses suggests that the cytosolic but not the mitochondrial isozyme has a hydrophobic patch or pocket capable of binding to the alkyl side chains of the affinity medium. 4. Evidence is presented to show that the cytosolic isozyme contains some basic amino acids whose ionizations are not expressed under native conditions. The mitochondrial isozyme does not show this effect. 5. Predictions of the secondary structures of the isozymes are presented based on four predictive models. Minimum predictions are derived on the basis of agreement of three out of four of the individual predictions. These aggregate predictions underestimate the amount of ordered secondary structure in both isozymes and suggest a relatively low degree of similarity between the isozymes. Individual predictive models, on the other hand, reveal greater similarities in secondary structures between the two isozymes. [ABSTRACT FROM AUTHOR]

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The confirmation of the syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three dimentional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported. [ABSTRACT FROM AUTHOR]

A copper, zinc superoxide dismutase, has been isolated from the marine turtle Caretta caretta and the complete amino acid sequence obtained. The sequence was determined by isolation and analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin or Staphylococcus aureus protease. Turtle superoxide dismutase consists of 166 amino acid residues, which represents the largest number to date for a cytosolic copper,zinc superoxide dismutase. The comparison of this amino acid sequence with that of bovine superoxide dismutase revealed a one-residue C-terminal extension, two single residue insertions and a 12-residue insertion in the N-terminal region, in turtle superoxide dismutase. The new segment consists of a threefold repeating sequence and was found to be the site for selective proteolytic attack by trypsin under native conditions. The biochemical characteristics, the spectroscopic and catalytic properties as well as the thermal stability and the resistance to irreversible denaturation, were carefully examined and were very similar to those of other superoxide dismutases. These results indicate that the presence of the new polypeptide segment does not affect the main folding of the chain and the quaternary structure, nor the functional parameters of turtle superoxide dismutase. The possibility that the new insert constitutes a loop excluded from the protein scaffold providing the framework of the active site is also discussed. [ABSTRACT FROM AUTHOR]

The copper,zinc-containing superoxide dismutase electromorphs from chicken erythrocytes have been isolated, their complete amino acid sequence determined and the identity of the protein moieties established. All electromorphs are constituted by a polypeptide chain made of 153 amino acid residues, corresponding to a molecular mass of 15598 Da. Accurate molecular mass determination by electrospray mass spectrometry of the separated electromorphs unequivocally proved that, in the chicken superoxide dismutase, either one or two cysteine residues/subunit are involved in a mixed disulfide with glutathione. The same post-translational modification has been proven to occur in human superoxide dismutase. A different rate of S-thiolation by endogenous glutathione was also demonstrated to be responsible for charge heterogeneity in cells. Effects of this modification on the catalytic and molecular properties of superoxide dismutases, and possible mechanisms for the S-thiolation process, were also investigated and discussed. [ABSTRACT FROM AUTHOR]

cDNA encoding human 4-aminobutyrate aminotransferase (aminobutyrate:2-oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu. J.-Y. & Tobin, A, J, (1994) J. Neurochem. 62. 1267–1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity o r the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme. [ABSTRACT FROM AUTHOR]

Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues. Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme-bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs. The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild-type enzyme. The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild-type enzyme and a 15-fold reduction in affinity for glycine and serine. The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate. Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro-aldol cleavage of serine ethyl ester and serinamide. The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild-type enzyme activity with serine, respectively. The R363A mutant enzyme catalyzed the half transamination of D-alanine methyl ester and L-alanine methyl ester at rates similar to the rates of transmination of D-al anine and L-alanine by the wild-type enzyme. The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase. Evidence is also provided that R363 may play a role in the substrate-induced open to closed conformational change of the active site. [ABSTRACT FROM AUTHOR]

The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215–7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107–142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli. [ABSTRACT FROM AUTHOR]

The amino acid sequence of glutamate decarboxylase from Escherichia coli was solved by a combination of automated Edman degradation of peptide fragments derived by proteolytic and chemical cleavage and sequencing of DNA. Correct alignment of three peptides, for which no peptide overlaps were available, was achieved by sequencing a 1.1-kbp fragment of DNA produced by a polymerase-chain reaction using primers corresponding to sequences known to be in amino-terminal and carboxy-terminal regions of the protein. Sequence similarity (24% identity) with mammalian glutamate decarboxylase was found to be limited to a 55-residue sequence around the lysine residue that binds the coenzyme. Stronger similarity (38% identity), again confined to the same region, is seen with bacterial pyridoxal-phosphate-dependent histidine decarboxylase. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage by trypsin, cyanogen bromide, Staphylococcus aureus V8 protease and pepsin, The enzyme subunit is composed of 421 amino acid residues yielding a molecular mass of 46.078 kDa, The presence of N-ε,-methyllysine in six positions of the sequence was observed. Comparison of the sequence of glutamate dehydrogenase from S. solfataricus with the other known primary structures of the corresponding enzyme from different sources, gives an overall identity of 9.2% and shows a symmetrical evolutionary distance of this archaebactcrial protein from the two groups of vertebrate on one side and eubacterial and low eucaryote enzymes on the other side. The occurrence of specific substitutions and a possible role for N-ε-methylation of lysine residues are discussed in view of current hypotheses on the molecular basis of thermal adaptation of proteins. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is reported. The sequence was determined from analysis of peptides isolated after tryptic and cyanogen bromide cleavages of the enzyme. Each subunit is made up of 485 residues, corresponding to a molecular mass of 53858 Da. The N- terminus of the polypeptide chain is an acetylated methionyl residue. A number of structural features, previously shown to be important for the structure and function of the enzyme, could be localized along the polypeptide chain. Comparison of the primary structure with the known cDNA-deduced sequences of other Dopa decarboxylases (i.e. the human, bovine, rat, guinea-pig and Drosophila enzymes) reveals 50% identity. The identity increases to 73%, if the comparison is restricted to the mammalian sequences. Comparison with other aromatic and nonaaromatic decarboxylases allows some consideration to be made in terms of structure/function and evolutionary relationships in this class of enzymes. [ABSTRACT FROM AUTHOR]

Homogeneous preparations of cytosolic serine hydroxymethyltransferase from rabbit liver were incubated with several different proteases. Chymotrypsin rapidly cleaves a tetradecapeptide from the NH2-terminal end of the enzyme with the enzyme retaining full catalytic activity. Trypsin digestion results in the release of several small peptides from the NH2-terminal end of the enzyme. The remaining core protein is reduced in molecular mass by about 3500 Da. With L-serine as substrate the core protein has 1.5 times the activity of the native enzyme. The difference in activity is due to a change in Vmax since the Km values for L-serine and tetrahydrofolate are unchanged. When allothreonine is used as the substrate the activity of the trypsin-treated enzyme is unchanged. Ks values for glycine and several folate compounds are also unchanged for the trypsin-digested enzyme. The relative distribution of three glycine-enzyme complexes shows only small differences between the native and trypsin-digested enzyme. Thermal denaturation studies show that the trypsin-digested enzyme has a thermal transition three degrees lower than the native enzyme but the same enthalpy of denaturation. These results suggest that the 25-30 amino acid residues from the NH2-terminal end of the enzyme are not important in determining the catalytic activity and structural stability of the purified enzyme. Several other proteases had no observable effect on the activity and size of the enzyme. All of the proteases tested inactivated the apoenzyme and digested it into small fragments. The loss of enzyme activity in frozen liver is probably the result of the enzyme slowly being converted to the apoenzyme form, which is susceptible to protease degradation. [ABSTRACT FROM AUTHOR]

The inactivation of aspartate aminotransferase in the course of the incubation with L-serine O-sulfate is protected by thiosulfate. It has been found that also L-cysteine sulfinic acid is an inactivator when it is incubated with the enzyme, while L-serine-O-phosphate and L-cysteine-S-sulfonate are inactive. The inactivation by L-cysteine sulfinic acid is increased by the simultaneous addition of 2-oxoglutrate and is protected by thiosulfate. Cysteine-S-sulfonate has been detected in the incubates of the enzyme with L-serine-O-sulfate protected with thiosulfate. It has been concluded that the protection by thiosulfate is due to the β-addition of thiosulfate to the inactivator aminoacrylic acid. The resulting cysteine-S-sulfonate thus represents an inactive dead-end product formed in the course of the protection by thiosulfate. It is suggested that this reaction could play the role of protecting enzymes sensitive to aminoacrylic acid and of inserting inorganic sulfur into aminoacrylic acid, producing a cysteine derivative. [ABSTRACT FROM AUTHOR]

Both holo- and apo-aspartate-aminotransferase are sensitive to tetranitromethane; however, the inactivation which follows nitration is partial for the holoenzyme, and complete for the apoenzyme. Cysteinyl and tyrosyl residues are modified in both forms of the enzyme; in the apoenzyme a greater number of tyrosines are available to the reagent. The complete inactivation of the apoenzyme is correlated with the loss of only one tyrosyl residue, which is selectively modified under particular conditions. The loss of activity is caused by tile failure of the nitrated apoenzyme to recombine with the coenzyme. [ABSTRACT FROM AUTHOR]