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PubMed1997

A nonsense mutation in the 3-hydroxy-3-methylglutaryl-CoA lyase gene produces exon skipping in two patients of different origin with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G-T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of acetone cyanohydrin lyase from flax (Linum usitatissimum). Definition of a novel class of hydroxynitrile lyases.

Acetone cyanohydrin lyase from Linum usitatissimum is a hydroxynitrile lyase (HNL) which is involved in the catabolism of cyanogenic glycosides in young seedlings of flax. We have isolated a full-length cDNA clone encoding L. usitatissimum HNL (LuHNL) from a cDNA expression library by immunoscreening. LuHNL cDNA was expressed in Escherichia coli and isolated from the respective soluble fraction in an active form which was biochemically indistinguishable from the natural enzyme. An open reading frame of 1266 base pairs encodes for a protein of 45,780 kDa. The derived amino acid sequence shows no overall homologies to the to date cloned HNLs, but has significant similarities to members of the alcohol dehydrogenase (ADH) family of enzymes. In particular, the cysteine and histidine residues responsible for coordination of an active site Zn2+ and a second structurally important Zn2+ in alcohol dehydrogenases are conserved. Nevertheless, we found neither alcohol dehydrogenase activity in LuHNL nor HNL activity in ADH. Moreover, well known inhibitors of ADHs, which interfere with the coordination of the active site Zn2+, fail to affect HNL activity of LuHNL, suggesting principally different mechanisms of cyanohydrin cleavage and alcohol oxidation. Interestingly, LuHNL like ADH and Prunus serotina (PsHNL) possesses an ADP-binding betaalphabeta unit motif, pointing to the possibility that the non-flavoprotein PsHNL and the flavoprotein LuHNL have developed from two independent lines of evolution of a common ancestor with an ADP-binding betaalphabeta unit.

Aldehyde-Lyases↗

PubMed1999

Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in nature.

Tyrosine phenol-lyase (TPL), which catalyzes the beta-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the beta-elimination activity toward dicarboxylic amino acids at least 10(4)-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine. beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid beta-lyase is an enzyme not found in nature.

Amino Acid Sequence↗

PubMed1990

Citrate lyase from Klebsiella pneumoniae. The complete primary structure of the acyl lyase subunit.

The primary structure of the beta-subunit (acyl lyase subunit) of citrate lyase from Klebsiella pneumoniae (ATCC 13,882) was determined with protein chemical methods. The polypeptide chain consists of 289 amino acid residues and has a molecular mass of 31,352 Da. The two half-cystine residues of the subunit are present as cysteines and not involved in disulfide bridges. The sequence shows no homology to known sequences of proteins or nucleic acids and reads (sequence; see text)

Amino Acid Sequence↗

PubMed2005

A gene encoding L-methionine gamma-lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine gamma-lyase.

Citrobacter freundii cells produce L-methionine gamma-lyase when grown on a medium containing L-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded L-methionine gamma-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3'-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.

Base Sequence↗

PubMed1998

Modeling study of some inhibitors of 17,20-lyase, a component of the enzyme 17 alpha-hydroxylase/17,20-lyase: a novel approach.

A novel molecular modeling study, involving inhibitors bound to a "substrate-heme complex," is described for the binding of steroidal and non-steroidal inhibitors of the 17,20-Lyase component of the enzyme complex 17 alpha-hydroxylase/17,20-lyase to gain further insight into the active site of this enzyme. This novel approach has resulted in the construction of a simple working model using which a number of compounds and their inhibitory activity have been rationalised.

Binding Sites↗

PubMed1984

Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties.

The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. Ei and the activating enzyme were purified to homogeneity and structurally characterized. Ei has an alpha 2 oligomeric structure (2 X 85 kDa) and contains no cofactor. The amino acid composition has been determined. Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k2 = 7 (M-1 s-1) at pH 6.8, 30 degrees C), which is accompanied by loss of the activatability of the enzyme. The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa. It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm. Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form Ea into Ei when anaerobic cells become nutrient-depleted.

Acetyltransferases↗

PubMed2004

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors.

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.

Animals↗

PubMed1981

Impaired induction and self-catabolite repression of extracellular pectate lyase in Erwinia chrysanthemi mutants deficient in oligogalacturonide lyase.

The pectate lyase (PL; EC 4.2.2.2) secreted by the plant pathogen Erwinia chrysanthemi is induced and catabolite repressed by different concentrations of its own product, digalacturonic acid 4,5-unsaturated at the nonreducing end [u(GalUA)(2)]. Both activities of u(GalUA)(2) depend on its cleavage by oligogalacturonide lyase (OGL; EC 4.2.2.6). This intracellular enzyme converts u(GalUA)(2) to the deoxyketuronic acid 4-deoxy-L-threo-5-hexosulose uronic acid, which is then isomerized to 3-deoxy-D-glycero-2,5-hexodiulosonic acid. An OGL-deficient mutant unable to grow on u(GalUA)(2) was poorly induced by u(GalUA)(2) or by D-galacturonan but produced wild-type levels of PL when supplied with 3-deoxy-D-glycero-2,5-hexodiulosonic acid. PL synthesis in the mutant could also be stimulated by 4,5-unsaturated trigalacturonic acid, from which deoxyketuronic acid is released by another intracellular enzyme. An OGL-deficient mutant that grew slowly on u(GalUA)(2) in comparison with the wild-type parent was hyperinduced by u(GalUA)(2) unless catabolite repression was relieved by cyclic AMP or imposed by logarithmic growth on glycerol. PL synthesis is also stimulated by saturated digalacturonic acid, which is released from D-galacturonan by another extracellular enzyme, exo-poly-alpha-D-galacturonosidase (EC 3.2.1.82). Because these dimers stimulate PL synthesis at concentrations (wt/vol) 1/1000th of the concentration required by D-galacturonan, and because an OGL-deficient mutant uninducible by dimers was also uninducible by D-galacturonan, we postulate that PL induction by pectic polymers entails extracellular formation of dimers and subsequent intracellular conversion to deoxyketuronic acids, the apparent inducers of PL.

Journal Article↗

PubMed1988

Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences.

The structural gene of pyruvate formate-lyase (pfl) and that of pyruvate-formate-lyase-activating enzyme were shown to be adjacent on the chromosomal map of Escherichia coli. DNA sequencing was performed along a stretch of 3592 nucleotides to obtain the amino acid sequences of both proteins. The derived primary structures (759 and 245 residues) were confirmed by partial structure analyses on the purified proteins. The open reading frames are separated by a 194-nucleotide stretch, and their flanking regions include signal elements that are compatible with separate control of protein synthesis from the two genes.

Acetyltransferases↗

PubMed2001

Investigations on inhibitors of human 17 alpha-hydroxylase-17,20-lyase and their interactions with the enzyme. Molecular modelling of 17 alpha-hydroxylase-17,20-lyase, Part II.

New methods in treatment of hormone-dependent diseases like prostate or breast cancer have become a major subject in medical and pharmaceutical research. Because of the direct correlation of cancer growth and hormone concentration, inhibition of hormone biosynthesis presents a promising strategy in cancer therapy. The key enzyme in androgen biosynthesis is the 17 alpha-hydroxylase-17,20-lyase a cytochrome P450 system, which specifically converts gestagens to androgens. Because the 3D-structure of the enzyme is still unknown most recently a ligand-based design was used to gain deeper insights into protein structure and function. In this paper we present molecular modelling studies on compounds acting as competitive inhibitors of the human 17 alpha-hydroxylase-17,20-lyase. The compounds developed by Hartmann et al. belong to two different structural classes and show a wide range of inhibitory potency. The physico-chemical properties of the molecules were investigated and compared by studying structural flexibility and by calculating molecular interactions fields. The superimposition of all inhibitors in a low energy conformation yielded in the common pharmacophore. In the second part of the paper individual inhibitors were docked into the active site of the enzyme model of CYP17 developed in our group. The dynamic behaviour and stability of the protein-inhibitor-complexes was studied. The protein ligand interactions observed in course of the molecular dynamics simulations correspond well with the experimental data.

Computer Simulation↗

PubMed1988

Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase.

The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed2001

Selective and sensitive detection of pectin lyase activity using a colorimetric test: application to the screening of microorganisms possessing pectin lyase activity.

Several methods have been described for the detection and quantification of polygalacturonase (PG) and pectin lyase (PL) activities. The most frequently used tests are the Nelson method using copper(II) and an arsenomolybdate reagent to detect PG activity, and the colorimetric method using thiobarbituric acid (TBA) to detect PL activity. We observed that none of these methods are suitable to differentiate between these two enzymatic activities. Therefore, we optimized the test conditions of the TBA method. As a result, the detection of the enzymatic beta-elimination (PL activity) became sensitive and selective. A basic pretreatment at 80 degrees C for 5 min of the solution which contains the pectin fragments of the PL activity furnished aldehydes which were condensed with TBA or its derivatives. After acidification of the medium, a pink fluorescent dye was detected spectrophotochemically (lambda = 550 nm). The interference of galacturonic acid or oligomers resulting from PG activity was completely eliminated. The most sensitive reagent was N-(pyridin-2-yl)-thiobarbituric acid. The application of this method with the new reagent was extended to the screening of microorganisms possessing the PL activity. The obtained results confirm that Aspergillus niger strain and a Saccharomyces cerevisiae SCPP strain possess this activity.

Aspergillus niger↗

PubMed2002

Purification and characterization of a cold-adapted isocitrate lyase and expression analysis of the cold-inducible isocitrate lyase gene from the psychrophilic bacterium Colwellia psychrerythraea.

Isocitrate lyase (ICL) from Colwellia psychrerythraea, a psychrophilic bacterium, was purified and characterized. The subunit molecular mass was 64 kDa, which is larger than that of other bacterial ICLs. The optimal temperature for its activity was 25 degrees C, the value of K(m) for the substrate ( DL-isocitrate) was minimum at 15 degrees C, and the catalytic efficiency ( k(cat)/ K(m)) value was maximum at 20 degrees C. Furthermore, the enzyme was remarkably thermolabile and completely inactivated by incubation for 2 min at 30 degrees C. These features indicate that ICL from this bacterium is a typical cold-adapted enzyme. A partial amino acid sequence of the C. psychrerythraea ICL was very similar to that of the closely related psychrophile Colwellia maris. Expression of the gene encoding the C. psychrerythraea ICL was found to be induced by low temperatures and by acetate in the medium. The cold adaptation of the catalytic properties of ICL and the stimulated expression of its gene at low temperatures strongly suggest that this enzyme is important for the growth of this bacterium in a cold environment.

Adaptation, Physiological↗

PubMed1988

Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Escherichia coli tryptophan indole-lyase.

Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV = 2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed2006

Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin beta-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin beta-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin beta-subunit.

Bacterial Proteins↗

PubMed1994

Gellan lyases--novel polysaccharide lyases.

A number of bacterial strains capable of degrading the bacterial exopolysaccharide gellan have been isolated by standard enrichment procedures. They include several pink-pigmented Gram-negative rod-shaped bacteria. A red-pigmented Gram-positive bacillus earlier found to degrade the exopolysaccharide xanthan from Xanthomonas campestris also showed slight gellanase activity. All the Gram-negative bacteria are non-fermentative, motile and amylase-producing. The gellan degradation in each case is due to eliminase-type enzymes (lyases) which appear to be extracellular enzymes cleaving the sequence... beta-D-glucosyl-(1-->4)-beta-D-glucuronosyl... in the tetrasaccharide repeat unit of the substrate polysaccharides. Although in some isolates these enzymes appear to be exo-acting, it appears from the loss in viscosity of the alternative substrate deacetylated rhamsan that they are predominantly endoenzymes. The enzyme activity is inducible: it is almost absent from glucose-grown cells. Associated with the 'gellanase' activity, all the Gram-negative bacterial isolates possess intracellular alpha-L-rhamnosidase and beta-D-glucosidase activities apparently located in the periplasm. The enzymes are highly specific and fail to cause significant degradation of most of the other bacterial exopolysaccharides which have been shown to be structurally related to gellan. As well as acting on gellan, they exert similar degradative activity against the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan. The enzymes only have relatively slight activity against the natural, acylated gellan-like polysaccharides from the bacteria now designated as strains of Sphingomonas paucimobilis.

Bacillus↗

PubMed1995

Use of Tn5tac1 to clone a pel gene encoding a highly alkaline, asparagine-rich pectate lyase isozyme from an Erwinia chrysanthemi EC16 mutant with deletions affecting the major pectate lyase isozymes.

Erwinia chrysanthemi mutant CUCPB5047, delta(pelA pelE) delta(pelB pelC)::28bp delta(pelX) delta 4bp pehX::omega Cmr, was constructed, mutated with Tn5tac1, and screened for isopropyl-beta-D-thiogalactopyranoside-dependent pectate lyase (Pel) production. A Kmr SacI fragment from the hyperexpressing Pel+ mutant CUCPB5066 was cloned into Escherichia coli and sequenced. The gene identified, pelL, encodes a novel, asparagine-rich, highly alkaline enzyme that is similar in primary structure to PelX and in enzymological properties to PelE.

Amino Acid Sequence↗