Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg(2+)

Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB). Here we report the X-ray crystal structure of the native and the pyruvate/Mg(2+) bound PrpB from S. typhimurium, determined at 2.1 and 2.3A, respectively. The structure closely resembles that of the Escherichia coli enzyme. Unlike the E. coli PrpB, Mg(2+) could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg(2+) was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg(2+) by 2.5A from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg(2+) bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.     

Crystal structure of 2-methylisocitrate lyase (PrpB) from Escherichia coli and modelling of its ligand bound active centre

Following acetate, propionate is the second most abundant low molecular mass carbon compound found in soil. Many microorganisms, including most, if not all fungi, as well as several aerobic bacteria, such as Escherichia coli and Salmonella enterica oxidize propionate via the methylcitrate cycle. The enzyme 2-methylisocitrate lyase (PrpB) from Escherichia coli catalysing the last step of this cycle, the cleavage of 2-methylisocitrate to pyruvate and succinate, was crystallised and its structure determined to a resolution of 1.9A. The enzyme, which strictly depends on Mg(2+) for catalysis, belongs to the isocitrate lyase protein family. A common feature of members of this enzyme family is the movement of a so-called "active site loop" from an open into a closed conformation upon substrate binding thus shielding the reactants from the surrounding solvent. Since in the presented structure, PrpB contains, apart from a Mg(2+), no ligand, the active site loop is found in an open conformation. This conformation, however, differs significantly from the open conformation present in the so far known structures of ligand-free isocitrate lyases. A possible impact of this observation with respect to the different responses of isocitrate lyases and PrpB upon treatment with the common inhibitor 3-bromopyruvate is discussed. Based on the structure of ligand-bound isocitrate lyase from Mycobacterium tuberculosis a model of the substrate-bound PrpB enzyme in its closed conformation was created which provides hints towards the substrate specificity of this enzyme.     

Structure of mandelate racemase with bound intermediate analogues benzohydroxamate and cupferron

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-? resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 ? between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.     

Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis

The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg(2+) ion.     

Crystal structure of the petal death protein from carnation flower

Expression of the PSR132 protein from Dianthus caryophyllus (carnation, clover pink) is induced in response to ethylene production associated with petal senescence, and thus the protein is named petal death protein (PDP). Recent work has established that despite the annotation of PDP in sequence databases as carboxyphosphoenolpyruvate mutase, the enzyme is actually a C-C bond cleaving lyase exhibiting a broad substrate profile. The crystal structure of PDP has been determined at 2.7 A resolution, revealing a dimer-of-dimers oligomeric association. Consistent with sequence homology, the overall alpha/beta barrel fold of PDP is the same as that of other isocitrate lyase/PEP mutase superfamily members, including a swapped eighth helix within a dimer. Moreover, Mg(2+) binds in the active site of PDP with a coordination pattern similar to that seen in other superfamily members. A compound, covalently bound to the catalytic residue, Cys144, was interpreted as a thiohemiacetal adduct resulting from the reaction of glutaraldehyde used to cross-link the crystals. The Cys144-carrying flexible loop that gates access to the active site is in the closed conformation. Models of bound substrates and comparison with the closed conformation of isocitrate lyase and 2-methylisocitrate lyase revealed the structural basis for the broad substrate profile of PDP.     

Diversity of function in the isocitrate lyase enzyme superfamily: the Dianthus caryophyllus petal death protein cleaves alpha-keto and alpha-hydroxycarboxylic acids

The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.     

Structural basis for the catalysis and substrate specificity of homoserine kinase

Homoserine kinase (HSK), the fourth enzyme in the aspartate pathway of amino acid biosynthesis, catalyzes the phosphorylation of L-homoserine (Hse) to L-homoserine phosphate, an intermediate in the production of L-threonine, L-isoleucine, and in higher plants, L-methionine. The high-resolution structures of Methanococcus jannaschii HSK ternary complexes with its amino acid substrate and ATP analogues have been determined by X-ray crystallography. These structures reveal the structural determinants of the tight and highly specific binding of Hse, which is coupled with local conformational changes that enforce the sequestration of the substrate. The delta-hydroxyl group of bound Hse is only 3.4 A away from the gamma-phosphate of the bound nucleotide, poised for the in-line attack at the gamma-phosphorus. The bound nucleotides are flexible at the triphosphate tail. Nevertheless, a Mg(2+) was located in one of the complexes that binds between the beta- and gamma-phosphates of the nucleotide with good ligand geometry and is coordinated by the side chain of Glu130. No strong nucleophile (base) can be located near the phosphoryl acceptor hydroxyl group. Therefore, we propose that the catalytic mechanism of HSK does not involve a catalytic base for activating the phosphoryl acceptor hydroxyl but instead is mediated via a transition state stabilization mechanism.     

Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg(2+) or Co(2+) being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7x10(-4)m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6x10(-5)m; acetyl-CoA, 1.5x10(-5)m; glyoxylate, 1.7x10(-3)m; Mg(2+), 1.2x10(-3)m.     

Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926

Chondroitin sulfate ABC lyase (ChonABC) is an enzyme with broad specificity that depolymerizes via beta-elimination chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs). ChonABC eliminates the glycosidic bond of its GAG substrates on the nonreducing end of their uronic acid component. This lyase possesses the unusual ability to act on both epimers of uronic acid, either glucuronic acid present in CS or iduronic acid in DS. Recently, we cloned, purified, and determined the three-dimensional structure of a broad specificity chondroitin sulfate ABC lyase from Bacteroides thetaiotaomicron (BactnABC) and identified two sets of catalytic residues. Here, we report the detailed biochemical characterization of BactnABC together with extensive site-directed mutagenesis resulting in characterization of the previously identified active site residues. BactnABC's catalysis is stimulated by Ca(2+) and Mg(2+) cations, particularly against DS. It displays extremely low activity toward hyaluronic acid and no activity toward heparin/heparan sulfate. Degradation of CS and DS by BactnABC yields only disaccharide products, pointing to an exolytic mode of action. The kinetic evaluations of the active-site mutants indicate that CS and DS substrates bind in the same active site, which is accompanied by a conformational change bringing the two sets of active site residues together. Conservative replacements of key residues suggest that His345 plays the role of a general base, initiating the degradation by abstracting the C5 bound proton from DS substrates, whereas either Tyr461 or His454 perform the equivalent role for CS substrates. Tyr461 is proposed, as well, to serve as general acid, completing the degradation of both CS and DS by protonating the leaving group.     

Structural insights into the Thermus thermophilus ADP-ribose pyrophosphatase mechanism via crystal structures with the bound substrate and metal

ADP-ribose pyrophosphatase (ADPRase) catalyzes the divalent metal ion-dependent hydrolysis of ADP-ribose to ribose 5'-phosphate and AMP. This enzyme plays a key role in regulating the intracellular ADP-ribose levels, and prevents nonenzymatic ADP-ribosylation. To elucidate the pyrophosphatase hydrolysis mechanism employed by this enzyme, structural changes occurring on binding of substrate, metal and product were investigated using crystal structures of ADPRase from an extreme thermophile, Thermus thermophilus HB8. Seven structures were determined, including that of the free enzyme, the Zn(2+)-bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADP-ribose and Zn(2+) or Gd(3+), and the product complexes with AMP and Mg(2+) or with ribose 5'-phosphate and Zn(2+). The structural and functional studies suggested that the ADP-ribose hydrolysis pathway consists of four reaction states: bound with metal (I), metal and substrate (II), metal and substrate in the transition state (III), and products (IV). In reaction state II, Glu-82 and Glu-70 abstract a proton from a water molecule. This water molecule is situated at an ideal position to carry out nucleophilic attack on the adenosyl phosphate, as it is 3.6 A away from the target phosphorus and almost in line with the scissile bond.     

Purification of the alliin lyase of garlic, Allium sativum L

1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at -10 degrees , particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6.5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10mum) and cysteine (0.5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The K(m) values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the K(m) was 6mm and the V(max.) was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The K(i) decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the K(i) was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg(2+), Mn(2+), Co(2+) or Fe(2+) stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg(2+).     

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase has been expressed in an active form in Escherichia coli and purified to homogeneity. Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system. After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg. The purified enzyme is stable when stored in 20% glycerol at -80 degrees C. The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins. The enzyme exhibits a Km = 20 microM for (S)-HMG-CoA. Divalent cations (Mg2+ and Mn2+) markedly stimulate the enzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans. The activator constant, K(a), for Mg2+ (6.9 mM) is 3 orders of magnitude greater than that for Mn2+ (2.0 microM). While EDTA does not affect activity, o-phenanthroline treatment markedly inhibits the enzyme. In contrast, m-phenanthroline is ineffective, suggesting that the ortho isomer's effect is attributable to chelation of a tightly bound metal ion. Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper. In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10. When the growth medium is supplemented with 1 mM CuSO4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit. Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis

The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure.     

Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.     

Briefly bound to activate: transient binding of a second catalytic magnesium activates the structure and dynamics of CDK2 kinase for catalysis

We have determined high-resolution crystal structures of a CDK2/Cyclin A transition state complex bound to ADP, substrate peptide, and MgF(3)(-). Compared to previous structures of active CDK2, the catalytic subunit of the kinase adopts a more closed conformation around the active site and now allows observation of a second Mg(2+) ion in the active site. Coupled with a strong [Mg(2+)] effect on in vitro kinase activity, the structures suggest that the transient binding of the second Mg(2+) ion is necessary to achieve maximum rate enhancement of the chemical reaction, and Mg(2+) concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the simultaneous binding of substrate peptide, ATP, and two Mg(2+) ions is able to induce a more rigid and closed organization of the active site that functions to orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated γ-phosphate from solvent.     

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: new insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/- 2 s(-1) and P-pyr Km = 5 +/- 1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/- 0.1 mM, 17 +/- 1 microM, and 210 +/- 10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.     

Biodegradation of all stereoisomers of the EDTA substitute iminodisuccinate by Agrobacterium tumefaciens BY6 requires an epimerase and a stereoselective C-N lyase

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe(2+)-, Mg(2+)-, and Ca(2+)-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn(2+)-IDS were rather scant and very slow on Cu(2+)-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.     

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.     

Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups.

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5'-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.