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PubMed1981

Properties of stearylamine liposomes containing tyrosine phenol-lyase.

The activity of tyrosine phenol-lyase, a chemotherapeutic enzyme with a dissociable pyridoxal phosphate cofactor, was studied after incorporation into multilamellar positively charged liposomes. Tyrosine phenol-lyase activity was assessed in the presence and absence of exogenous pyridoxal phosphate. A maximum of 75% total enzyme activity was associated with liposomes when prepared from a molar lipid ratio of egg lecithin, cholesterol, stearylamine (7 : 2 : 1, w/w). The total tyrosine phenol-lyase activity was comprised of 25% membrane-associated enzyme and 50% encapsulated enzyme. Encapsulation increased the stability of the enzyme under the in vitro conditions of cold storage at 4 degrees C for 3 weeks and under elevated temperatures up to 61 degrees C. Liposomal encapsulation afforded little protection against trypsin and no protection against whole mouse plasma in vitro. Heat-treated plasma (100 degrees C for 1 h) had little effect on the activity of free and encapsulated tyrosine phenol-lyase. These results indicated that whole plasma contained a heat-labile factor(s) which destroyed both the liposomal and free tyrosine phenol-lyase activity. Plasma clearance after intraperitoneal injection of tyrosine phenol-lyase in B6D2F1 female mice was retarded by liposomal encapsulation, particularly when the animals were pre-treated with empty liposomes; however, only a small proportion of free and liposomal tyrosine phenol-lyase was absorbed. The free enzyme rapidly lost holoenzyme activity after absorption but the liposomes maintained holoenzyme activity. Even though liposomes preserved holo-tyrosine phenol-lyase activity, the holoenzyme was not present in sufficient concentration to sustain a reduced plasma tyrosine level.

Amines↗
PubMed1978

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.

Acetates↗
PubMed1998

Human DNA polymerase beta deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism.

DNA polymerase beta (beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of beta-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.

Carbon-Oxygen Lyases↗
PubMed1984

Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase.

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.

Acetates↗
PubMed1986

Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene.

Bacterial Outer Membrane Proteins↗
PubMed1997

Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70 degrees C with a growth rate of 0.23 h(-1) with pectin and 0.12 h(-1) with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70 degrees - 75 degrees C), amylolytic as well as pullulolytic enzymes (highest activity at 80 degrees - 85 degrees C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135000 Da whereas lyase b consisted of two subunits with molecular masses of 93000 Da and 158000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80 degrees C. The enzymes were very stable at 70 degrees C and at 80 degrees C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The Km values of both lyases for pectate and citrus pectin were 0.5 g(-1) and 5.0 g(-1), respectively. After incubation with polygalacturonic acid, mono-, di-, and trigalacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca2+. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov.

Bacteria, Anaerobic↗
PubMed1992

Regulation of ATP-citrate lyase at transcriptional and post-transcriptional levels in rat liver.

The amounts of ATP-citrate lyase in liver cytosol began to increase at 12 hours after refeeding a high-carbohydrate diet and further increased until 48 hours. The amounts of the ATP-citrate lyase mRNA began to increase at 6 hours and reached to a maximum level at 12 hours, followed by decrease to a very low level until 48 hours. The elevated amount of the ATP-citrate lyase mRNA reflected on the increase of ATP-citrate lyase content in the first 24 hours, but these two parameters were not paralleled thereafter. The transcriptional activity of ATP-citrate lyase gene in nuclei of rat liver began to increase at 4 hours and further increased to reach a maximum level of 24 fold at 12 hours, maintaining a high level of 17 fold until 48 hours. The elevation of transcriptional activity of ATP-citrate lyase gene preceded the increase of ATP-citrate lyase mRNA content in the liver cytosol by 2 hours, and its increasing pattern was similar to changes of mRNA content until 12 hours. However, while the transcriptional activity remained at a high level until 48 hours, the ATP-citrate lyase mRNA concentration in the cytosol decreased after 12 hours.

ATP Citrate (pro-S)-Lyase↗
PubMed1999

alpha-1,4-glucan lyases producing 1,5-anhydro-D-fructose from starch and glycogen have sequence similarity to alpha-glucosidases.

In the past few years a novel enzyme alpha-1,4-glucan lyase (EC 4.2. 2.13), which releases 1,5-anhydrofructose from starch and glycogen, has been cloned and characterized from red algae and fungi. Accumulated evidence indicates that the lytic degradation of starch and glycogen also occurs in other organisms. The present review focuses on the biochemical and molecular aspects of eight known alpha-1,4-glucan lyases and their genes from red algae and fungi. While the amino acid sequence identity is 75-80% among the alpha-1, 4-glucan lyases from each of the taxonomic groups, the identity between the algal and fungal alpha-1,4-glucan lyases is only 25-28%. Notably database searches disclosed that the alpha-1,4-glucan lyases have a clear identity of 23-28% with alpha-glucosidases of glycoside hydrolase family 31, thus for the first time linking enzymes from the class of hydrolases with that of lyases. The alignment of lyases and alpha-glucosidases revealed seven well-conserved regions, three of which have been reported to be involved in catalysis and substrate binding in alpha-glucosidases. The shared substrate and inhibitor specificity and sequence similarity of alpha-1,4-glucan lyases with alpha-glucosidases suggest that related structural elements are involved in the two different catalytic mechanisms.

Amino Acid Sequence↗
PubMed1995

Kinetic studies of cytochrome P-45017 alpha, lyase dependent androstenedione formation from progesterone.

The reaction mechanism of androstenedione formation from progesterone was analyzed in a membrane reconstituted system consisting of P-45017 alpha, lyase and NADPH-cytochrome P-450 reductase using a rapid quenching device at 10 degrees C. In these rapid quenching experiments, only the metabolites of [3H]progesterone bound to P-45017 alpha, lyase at the initial stage were detectable during the limited cycles of the P-45017 alpha, lyase reactions (1-120 s). The level of 17 alpha-hydroxy[3H]progesterone increased rapidly in a short period (1-5 s) and then decreased to about half. That of [3H]androstenedione increased gradually from 2 s, which exactly corresponded to the decrease in 17 alpha-hydroxy[3H]progesterone. 17 alpha-Hydroxyprogesterone was conclusively the actual intermediate steroid which did not dissociate from P-45017 alpha, lyase during the successive hydroxylation reaction into androstenedione. A kinetic model can clearly describe the successive reaction catalyzed by P-450 17 alpha, lyase, in which progesterone is converted successively into androstenedione via 17 alpha-hydroxyprogesterone, some of which dissociates from the active site of P-450 17 alpha, lyase and is never metabolized into androstenedione. We analyzed the effects of pH and the amount of NADPH-cytochrome P-450 reductase on the successive reaction and proved that the reaction was regulated by the rate of electron transfer for the conversion of the bound 17 alpha-hydroxyprogesterone to androstenedione. Furthermore, we found that the product dissociation from P-450 17 alpha, lyase is the rate-limiting process in the steady-state metabolism of progesterone by P-450 17 alpha, lyase.

17-alpha-Hydroxyprogesterone↗
PubMed1997

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99.

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.

Amino Acid Sequence↗
PubMed2000

Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics.

Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases.

Aldehyde-Lyases↗
PubMed2001

2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes.

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.

Amino Acid Sequence↗
PubMed1996

Modeling of a mutation responsible for human 3-hydroxy-3-methylglutaryl-CoA lyase deficiency implicates histidine 233 as an active site residue.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude. Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.

Acyl Coenzyme A↗
PubMed1998

Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase beta as revealed by site-directed mutagenesis. DNA binding and 5'-deoxyribose phosphate lyase activities.

The amino-terminal 8-kDa domain of DNA polymerase beta functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5'-phosphate recognition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive. The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72 as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.

Catalysis↗
PubMed1995

Structural analysis of the L-methionine gamma-lyase gene from Pseudomonas putida.

The gene encoding L-methionine gamma-lyase from Pseudomonas putida was cloned and the primary structure of the enzyme was deduced from its nucleotide sequence. The L-methionine gamma-lyase gene was expressed in Escherichia coli. The amino acid sequences of BrCN-digested peptides agreed with the corresponding parts of the L-methionine gamma-lyase sequence determined from the gene structure. The polypeptide is composed of 398 amino acid residues with a calculated molecular weight of 42,626, corresponding to the subunit of the homotetrameric enzyme. The deduced amino acid sequence of L-methionine gamma-lyase only showed extensive homology with other well known alpha,gamma-elimination and/or gamma-replacement pyridoxal 5'-phosphate-dependent enzymes, such as cystathionine gamma-lyase, cystathionine gamma-synthase, and O-acetylhomoserine O-acetylserine sulfhydrylase, that participate in the biosynthesis of sulfur amino acids. However, the deduced essential cysteine residue of L-methionine gamma-lyase was not conserved in these enzymes. We confirmed the presence of a part of an open reading frame in the 3'-flanking region of the L-methionine gamma-lyase gene, which showed high homology with the N-terminal region of pyruvate dehydrogenase (lipoamide) from E. coli, suggesting that it participates in the degradative pathway for L-methionine together with L-methionine gamma-lyase.

Amino Acid Sequence↗
PubMed2002

Structure of pectate lyase A: comparison to other isoforms.

Pectate lyase A is a virulence factor secreted by the plant-pathogenic bacteria Erwinia chrysanthemi. The enzyme cleaves the glycosidic bond of pectate polymers by a calcium-dependent beta-elimination mechanism. The crystal structure of pectate lyase A from E. chrysanthemi EC16 has been determined in two crystal forms, monoclinic C2 to 1.8 A and rhombohedral R3 to 2.1 A. The protein structure is compared with two other pectate lyase isoforms from E. chrysanthemi EC16, pectate lyase C and pectate lyase E. Pectate lyase A is unique as it is the only acidic pectate lyase and has end products that are significantly more varied in length in comparison to those of the other four major pectate lyase isozymes. Differences and similarities in polypeptide trace, size and volume of the active-site groove and surface electrostatics are discussed.

Binding Sites↗
PubMed2004

Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase.

In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.

Amino Acid Sequence↗
PubMed1977

Purification and properties of citrate lyase ligase from Streptococcus diacetilactis.

Citrate lyase ligase (acetate: SH--[acyl-carrier protein] enzyme ligase (AMP) from Streptococcus diacetilactis was purified 920-fold with a yield of 6.3%. The molecular weight of the enzyme was estimated to be 41000; the ligase consisted of one polypeptide chain. The acetylation of 1 mol of deacetyl-citrate lyase to enzymatically active citrate lyase required 6 mol ATP. The formation of AMP and pyrophosphate in the acetylation reaction was demonstrated. Citrate lyase ligase was specific for the lyase from S. diacetilacitis and did not acetylate lyases from Rhodopseudomonas gelatinosa and Enterobacter aerogenes. The substract acetate and ATP could be replaced by propionate and dATP, repectively. The reaction rates for ATP, acetate and deacetyl-citrate lyase followed Michaelis-Menten kinetics (Km values: 26 micron for ATP, 25 mM for acetate and 38 nM for deacetyl-citrate lyase).

Citrates↗