Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

Biochemical and Pharmacokinetic Properties of PEGylated Cystathionine γ-Lyase from Aspergillus carneus KF723837.

Cystathionine γ-lyase (CGL) was purified to its electrophoretic homogeneity from Aspergillus carneus by various chromatographic approaches. The purified enzyme has four identical subunits of 52 kDa based on SDS and native PAGE analyses. To improve its structural stability, purified CGL was modified by covalent binding to polyethylene glycol moieties. The specific activity of free-CGL and PEG-CGL was 59.71 and 48.71 U/mg, respectively, with a PEGylation yield of 81.5 and 70.7% modification of surface ε-amino groups. Free- and modified CGL have the same pattern of pH stability (8.0-9.0). At 50 °C, the thermal stability [half-life time (T1/2)] of PEG-CGL was increased by 40% in comparison to free-CGL. The activity of CGL was completely inhibited by hydroxylamine and Hg(+2), with no effect by EDTA. Free-CGL (0.04 mM(-1)s(-1)) and PEG-CGL (0.03 mM(-1)s(-1)) have a similar catalytic efficiency to L-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µM for the free- and PEG-CGL, respectively. By in vitro proteolysis, PEG-CGL retains >50% of its initial activity compared to <10% of the free-CGL for acid protease for 30 min. From in vivo pharmacokinetics in New Zealand white rabbits, the T1/2 was 19.1 and 28.9 h for the Holo free-CGL and PEG-CGL, respectively, ensuring the role of PEGylation on shielding the CGL surface from proteolytic attack, reducing its antigenicity, and stabilizing its internal Schiff base. By external infusion of pyridoxal 5'-phosphate (10 µM), the T1/2 of free- and PEG-CGL was prolonged to 24 and 33 h, respectively, so dissociation of pyridoxal 5'-phosphate was one of the main causes of loss of enzyme activity. The biochemical and hematological responses of rabbits to free- and PEG-CGL were assessed, with relative similarity to the negative control, confirming the nil toxicity of enzymes. The titer of IgG was duplicated in response to free- versus PEG-CGL after 45 days. To the best of our knowledge, this is the first report concerned with purification and PEGylation of CGL from fungi, with higher affinity for L-cystathionine. With further molecular studies, CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H2S).

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source.

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full-length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l-cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l-cystine and some other cysteine conjugates, but not l-cystathionine or l-methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l-cysteine as a nitrogen source, and that overexpression of the gene results in increased H2 S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S-ethyl-l-cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis.

Analysis of some enzymes activities of hydrogen sulfide metabolism in plants.

Hydrogen sulfide (H2S) which is considered as a novel gasotransmitter after reactive oxygen species and nitric oxide in plants has dual character, that is, toxicity that inhibits cytochrome oxidase at high concentration and as signal molecule which is involved in plant growth, development, and the acquisition of tolerance to adverse environments such as extreme temperature, drought, salt, and heavy metal stress at low concentration. Therefore, H2S homeostasis is very important in plant cells. The level of H2S in plant cells is regulated by its synthetic and degradative enzymes, L-/D-cysteine desulfhydrase (L-/D-DES), sulfite reductase (SiR), and cyanoalanine synthase (CAS), which are responsible for H2S synthesis, while cysteine synthase (CS) takes charge of the degradation of H2S, but its reverse reaction also can produce H2S. Here, after crude enzyme is extracted from plant tissues, the activities of L-/D-DES, SiR, CAS, and CS are measured by spectrophotometry, the aim is to further understand homeostasis of H2S in plant cells and its potential mechanisms.

Identification and characterization of a strain-dependent cystathionine beta/gamma-lyase in Lactobacillus casei potentially involved in cysteine biosynthesis.

The trans-sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans-sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans-sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei, which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli. The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds.

Interconversion of a pair of active-site residues in Escherichia coli cystathionine gamma-synthase, E. coli cystathionine beta-lyase, and Saccharomyces cerevisiae cystathionine gamma-lyase and development of tools for the investigation of their mechanisms and reaction specificity.

Cystathionine gamma-synthase (CGS) and cystathionine beta-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine gamma-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share approximately 30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/KmL-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/KmL-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The DeltametB and DeltametC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme.

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.

Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine gamma-lyase-like protein from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine gamma-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 A resolution and belonged to the primitive orthogonal space group P2(1)2(1)2(1) and the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 A and a = b = 78.2, c = 300.7 A, respectively. For the P2(1)2(1)2(1) crystals, three or four monomers exist in the asymmetric unit with a corresponding V(M) of 3.02 or 2.26 A(3) Da(-1) and a solvent content of 59.3 or 45.7%. For the P4(1) (or P4(3)) crystals, four or five monomers exist in the asymmetric unit with a corresponding V(M) of 2.59 or 2.09 A(3) Da(-1) and a solvent content of 52.5 or 40.6%.

One-step elimination of L-cysteine desulfhydrase from crude enzyme extracts of Pseudomonas sp. TS1138 using an immunomagnetic affinity matrix improves the enzymatic production of L-cysteine.

In this study, a high efficiency immunomagnetic affinity matrix was developed to eliminate L-cysteine desulfhydrase (CD), which decomposes L-cysteine, in crude enzyme extracts from Pseudomonas sp. TS1138. After cloning and expression in Escherichia coli, recombinant CD was purified to raise polyclonal antibodies from mice. The anti-CD antibody was cross-linked to staphylococcal protein A-magnetic cellulose microspheres (MCMS) with dimethyl pimelimidate (DMP). The natural CD was eliminated from the crude enzyme extracts by treatment with the cross-linked antibody-protein A-MCMS, resulting in a high level of L-cysteine production. The conversion rate of DL-2-amino-Delta2-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine increased significantly from 61.9 to 96.2%. The cross-linked antibody-protein A-MCMS showed its durability after repetitive use, maintaining a constant binding capacity for CD during five cycles. This study may lead to a convenient and cost-efficient method to produce L-cysteine by enzymatic conversions.

L-cysteine desulfidase: an [4Fe-4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia, and sulfide.

A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being involved in the formation of the [4Fe-4S] center and the thiolate of Cys25 serving as a base to abstract the alpha-hydrogen in the first step of the elimination. Although the enzyme has no sequence homology to any known enzymes, including the non-PLP-dependent serine/threonine dehydratases or aconitases, the mechanisms of action of all of these enzymes are similar, in that each catalyzes an alpha,beta-elimination reaction adjacent to a carboxylate group. It is proposed that the enzyme may be responsible for the production of sulfide required for the biosynthesis of iron-sulfur centers in this archaea. A mechanism of action of the enzyme is proposed.

Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana.

In several organisms D-cysteine desulfhydrase (D-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes D-cysteine into pyruvate, H2S, and NH3. A gene encoding a putative D-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has D-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5'-phosphate binding site. The purified recombinant mature protein had a Km for D-cysteine of 0.25 mm. Only D-cysteine but not L-cysteine was converted by D-CDes to pyruvate, H2S, and NH3. The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5'-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded D-CDes protein in the mitochondria. D-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; D-CDes protein levels remained almost unchanged in the same plants whereas specific D-CDes activity was highest in senescent plants. In plants grown in a 12-h light/12-h dark rhythm D-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of D-CDes RNA and increased protein levels, the D-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsisd-CDes protein are discussed.

Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production.

We highly purified the enzyme having L-cysteine desulfhydrase activity from Corynebacterium glutamicum. According to its partial amino acid sequence, the enzyme was identified as the aecD gene product, a C-S lyase with alpha, beta-elimination activity [I. Rossol and A. Pühler (1992) J. Bacteriol. 174, 2968-2977]. To produce L-cysteine in C. glutamicum, the Escherichia coli-altered cysE gene encoding Met256Ile mutant serine acetyltransferase, which is desensitized to feedback inhibition by L-cysteine, was introduced into C. glutamicum. When the altered cysE gene was expressed in the aecD-disrupted strain, the transformants produced approximately 290 mg of L-cysteine plus L-cystine per liter from glucose. The produced amount of these amino acids was about two-fold higher than that of the wild-type strain.

Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum.

Hydrogen sulfide and methyl mercaptan are the two major compounds associated with oral malodor. These compounds are highly toxic, and are thought to play an important role in periodontal disease. Fusobacterium nucleatum, an oral bacterium, produces large amounts of hydrogen sulfide from L-cysteine by the enzymatic action of L-cysteine desulfhydrase. We cloned and sequenced the cdl gene encoding L-cysteine desulfhydrase from F. nucleatum ATCC 10953, and revealed that the structural cdl gene consists of 921 bp and encodes a 33.4-kDa protein. The cloned gene was inserted into an expression vector, pDEST17, and expressed in Escherichia coli as a fused protein. The purified enzyme was tested for substrate specificity using various SH-containing compounds. Only L-cysteine served as a substrate for L-cysteine desulfhydrase to produce hydrogen sulfide.

Lactobacillus reuteri DSM 20016: purification and characterization of a cystathionine gamma-lyase and use as adjunct starter in cheesemaking.

A homo-tetrameric approximately 160-kDa cystathionine gamma-lyase was purified to homogeneity from Lactobacillus reuteri DSM 20016 by four chromatographic steps. The activity was pyridoxal-5'-phosphate dependent and the enzyme catalyzed the alpha,gamma-elimination reaction of L-cystathionine, producing L-cysteine, ammonia and alpha-ketobutyrate. The enzyme was active towards a range of amino acids and amino acid derivatives, including methionine. The pH and temperature optima were found to be 8.0 and 35 degrees C. respectively. Isoelectric pH (pI) was approximately 5.0 as determined by two-dimensional electrophoresis. Sensitivity to chemical inhibitors was typical of lactococcal cystathionine gamma- and beta-lyases, except it was inhibited by sulphydryl reagents. The N-terminal sequence was MKFNTQLIHGGNSED, which had 100% homology with cystathionine beta-lyase of Lb. reuteri 104R (Accession Number (CAC05298). Lb. reuteri DSM 20016. together with 10 other strains of non-starter lactic acid bacteria, was used as adjunct starter in the production of miniature ('anestrato Pugliese-like cheeses. After 40 d ripening, the water-soluble extract of the cheeses with added Lactobacillus fermentum DT41 and Lb. reuteri DSM 20016 contained the highest enzyme activities on cystathionine and methionine substrates. Determinations of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide in the miniature cheeses confirmed the findings of enzyme activities.

Spectroscopic and kinetic analyses reveal the pyridoxal 5'-phosphate binding mode and the catalytic features of Treponema denticola cystalysin.

To obtain insight into the functional properties of Treponema denticola cystalysin, we have analyzed the pH- and ligand-induced spectral transitions, the pH dependence of the kinetic parameters, and the substrate specificity of the purified enzyme. The absorption spectrum of cystalysin has maxima at 418 and 320 nm. The 320 nm band increases at high pH, while the 418 nm band decreases; the apparent pK(spec) of this spectral transition is about 8.4. Cystalysin emitted fluorescence at 367 and 504 nm upon excitation at 320 and 418 nm, respectively. The pH profile for the 367 nm emission intensity increases above a single pK of approximately 8.4. On this basis, the 418 and 320 nm absorbances have been attributed to the ketoenamine and substituted aldamine, respectively. The pH dependence of both log k(cat) and log k(cat)/K(m) for alpha,beta-elimination reaction indicates that a single ionizing group with a pK value of approximately 6.6 must be unprotonated to achieve maximum velocity. This implies that cystalysin is more catalytically competent in alkaline solution where a remarkable portion of its coenzyme exists as inactive aldamine structure. Binding of substrates or substrate analogues to the enzyme over the pH range 6-9.5 converts both the 418 and 320 nm bands into an absorbing band at 429 nm, assigned to the external aldimine in the ketoenamine form. All these data suggest that the equilibrium from the inactive aldamine form of the coenzyme shifts to the active ketoenamine form on substrate binding. In addition, reinvestigation of the substrate spectrum of alpha,beta-elimination indicates that cystalysin is a cyst(e)ine C-S lyase rather than a cysteine desulfhydrase as claimed previously.

A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors.

MalT, the transcriptional activator of the Escherichia coli maltose regulon, self-associates, binds promoter DNA and activates initiation of transcription only in the presence of ATP and maltotriose, the inducer. In vivo studies have revealed that MalT action is negatively controlled by the MalY protein. Using a biochemical approach, we analyse here the mechanism whereby MalY represses MalT activity. We show that MalY inhibits transcription activation by MalT in a purified transcription system. In vitro, a constitutive MalT variant (which is partially active in the absence of maltotriose) is less sensitive than wild-type MalT to repression by MalY, as observed in vivo. We demonstrate that MalY forms a complex with MalT only in the absence of maltotriose and that, conversely, MalY inhibits maltotriose binding by MalT. Together, these results establish that MalY acts directly upon MalT without the help of any factor, and that MalY is a negative effector of MalT competing with the inducer for MalT binding.

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363.

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.

Cystalysin, a 46-kDa L-cysteine desulfhydrase from Treponema denticola: biochemical and biophysical characterization.

A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.

Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica).

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.

Purification and characterization of cystathionine gamma-lyase from Lactobacillus fermentum DT41.

A homo-tetrameric ca. 140-kDa cystathionine gamma-lyase was purified to homogeneity from Lactobacillus fermentum DT41 by four chromatographic steps. This was the first enzyme responsible for amino acid catabolism purified from lactobacilli. The activity is pyridoxal-5'-phosphate dependent and the enzyme catalyzes the alpha,gamma-elimination reaction of L-cystathionine producing L-cysteine, ammonia and alpha-ketobutyrate. The cystathionine gamma-lyase produced a free thiol group, a keto acid component and ammonia from several amino acids, including L-cysteine and methionine, and amino acid derivatives. L-Cystine was the best substrate. The enzyme was stable in the conditions of cheese ripening and may contribute to the biosynthesis of sulfur-containing compounds.