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442 Cystalysin, a 46-kDa L-Cysteine Desulfhydrase from Treponema denticola: Biochemical and Biophysical Characterization Lianrui Chu, Jeffery L. Ebersole, Gary P. Kurzban, and Stanley C. Holt From the Departments of Microbiology and Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas 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 b-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (Na-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-phosphatecontaining enzyme with the activity of an aC-N and bC-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. Increasing evidence indicates that the oral treponemes play a significant role in the microbial ecology associated with the destructive events of periodontal disease [1 – 5]. Riviere et al. [6, 7] have described pathogen-related oral spirochetes (PROS), some of which are believed to be similar to Treponema vincentii [8], and Simonson and his co-workers [5, 9, 10] and Riviere et al. [6, 7, 11, 12] have also documented the importance of the oral treponemes in the progression of periodontal disease. In vitro, Treponema denticola produces a large number of purported virulence factors, including tissue-degrading enzymes, cytotoxic factors [13 – 21], and at least two types of proteins, which interact with selected host cells [22 – 24]. These factors, if functional in the in vivo environment of the host, could contribute significantly to the bone and tissue destruction characteristic of this inflammatory oral disease. Several reports [25 – 27] have described H2S formation as an end product of the metabolism of human serum proteins, as well as from cysteine and glutathione. H2S has also been found in relatively high levels (ú2 mM) in periodontal disease pockets. Since H2S is highly toxic for mammalian cells [28, 29], the enzymes participating in the production of H2S in the septic subgingival sulcus might contribute to the initiation and This article is part of a series of papers presented at a symposium entitled ‘‘Molecular Mechanisms of Microbial Host Cell Interactions in Periodontal Disease’’ that was held on 14 – 18 March 1997 in St. Petersburg, Florida. Grant support: grants DE-11368-03, DE-11771, and DE 07263. Portions of this work have been accepted for publication in Infection and Immunity. Reprints or correspondence: Stanley C. Holt, Department of Microbiology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7758. Clinical Infectious Diseases 1999;28:442–50 q 1999 by the Infectious Diseases Society of America. All rights reserved. 1058–4838/2803–0004$03.00 / 9c63$$mr30 02-24-99 10:52:23 progression of periodontal diseases [25 – 27]. Only a limited number of oral bacterial species have the metabolic capabilities necessary to produce and survive in the presence of high levels of H2S [26, 27], and T. denticola is a prominent representative of this physiological phenotype. Chu et al. [30 – 32] have recently described the isolation of a 46-kDa protein (cystalysin) from T. denticola that possesses both hemoxidative and hemolytic activities and is regulated by the amount of iron in the in vitro growth medium. The gene encoding this protein was cloned, sequenced, and expressed in Escherichia coli [33], and the amino acid sequence deduced by DNA sequencing of cystalysin exhibited significant identity to a family of aminotransferases [33, 34] with either aspartate aminotransferase [35, 36] or bC-S lyase activities [37, 38]. The study presented here describes the enzymatic activity of the 46-kDa cystalysin as a cysteine desulfhydrase and postulates a mechanism by which this protein could function in vivo as a virulence factor. Materials and Methods Bacterial Strains and Growth Condition T. denticola ATCC 35404 (TD-4) was grown as previously described [39]. Since iron-starvation resulted in an upregulation of cystalysin [30], we incorporated 200 mmol of BPD (2,2bipyridyl) into 1 liter of GM-1-cysteine medium for the purification of cystalysin. The E. coli recombinant strain carrying plasmid pLC67 [33] (E. coli LC-67), was used to isolate the recombinant 46-kDa protein. E. coli LC-67 was grown in LB broth as described by Chu et al. [33]. The cells were harvested during exponential growth by centrifugation at 9,000g for 10 minutes. Pellets of both T. denticola TD-4 and E. coli LC-67 were suspended in 10 mM of PBS (pH, 7.5), washed twice, and cida UC: CID CID 1999;28 (March) Cysteine Desulfhydrase from T. denticola resuspended in the same buffer. A proteinase inhibitor cocktail [40] was added to the cells, and the suspension was mixed thoroughly and stored frozen at 0207C until used. Fractionation and Purification of the 46-kDa Protein of T. denticola TD-4 The purification of the native cystalysin from T. denticola TD-4 was carried out as described by Chu and Holt [31]. The recombinant 46-kDa protein was purified as described below. Initial enrichment of the 46-kDa protein. Frozen E. coli LC-67 was thawed slowly and diluted with PBS to a whole-cell protein concentration of 4.5 mg/mL. The cells were sonicated (Branson Cell Disrupter 200; Sonic Power, Danbury, CT) for 10 minutes at maximum output at 47C, and the soluble fraction was separated by ultracentrifugation at 150,000g for 2 hours. This soluble cell fraction was used for the isolation of cystalysin by solid (70% to 90%) (NH4)2SO4 (AS) precipitation. DEAE-sephacel chromatography. Approximately 15 mL of a 70% – 90% AS fraction was applied to a DEAE-sephacel column (2.5 1 14 cm; Pharmacia Fine Chemicals, Uppsala, Sweden), equilibrated with 10 mM of phosphate buffer (pH, 7.5). Protein fractions were eluted from the column, and the fractions were collected and assayed for protein concentration and hemoxidative (HeO), hemolytic (HeA), and H2S activity (see below). Fractions with high activity were pooled, dialyzed (see above), and concentrated (see below; referred to as DEAEF). Preparative electrophoresis. The cystalysin was purified from the DEAEF by two rounds of preparative electrophoresis (Model 491 Prep Cell; Bio-Rad, Richmond, CA). Fractions were collected at an elution rate of 50 mL/h. Protein, HeA, HeO, and H2S activities were determined for each fraction. Based on the enzyme activity, the fractions containing §15 mg of protein/mL were concentrated by speed vacuum concentration (SpeedVac Concentrator [SC 100]; Savant, Farmingdale, NY), combined, and dialyzed. Protein concentration was determined by the Bio-Rad protein assay [41]. 443 Amino Acid Composition and Analysis Cystalysin from either T. denticola TD-4 or E. coli LC-67 (40 mg) was electrophoresed through SDS – 7.5% polyacrylamide gels, and the purified protein band was transferred to Immobilon P membranes (Millipore, Marlborough, MA) [23] and subjected to amino acid analysis on a Beckman System 7300 amino acid analyzer (Beckman, Brea, CA) [23]. Enzyme and End-Product Analysis Cysteine desulfhydrase activity was routinely assayed by quantifying H2S (see below and [20]). Unless otherwise indicated, all assays were performed in PBS (pH, 7.5). Hydrogen sulfide. Sulfide formation was quantitated as described by Siegel [43] except that the reaction mixture was prepared in a 1-mL volume in a 1.5-mL microcentrifuge tube sealed with a parafilm wrapped cap. Sulfide concentration was determined from a Na2S standard curve. Pyruvic acid analysis. Pyruvate formation was analyzed as described by Zheng et al. [44], with some modification. In brief, the reaction mixture consisted of cystalysin and the substrates (cysteine or cystathionine or others) to be studied and was incubated for 15 minutes at 377C. A 0.25-mL aliquot of the resulting supernatants was mixed with 0.25 mL each of 1.5 M Tris-HCl (pH, 7.5), 0.58 mM NADH, and distilled H2O. Twenty units of lactate dehydrogenase (20 mL) was added, and the oxidation of NADH was recorded on a DU-65 spectrophotometer (Beckman) as the decrease in absorbance at 340 nm, compared with a pyruvic acid standard curve. Ammonia. NH3 formation was assayed by modification of the method of Bauer et al. [45]. The NH3 concentration was calculated from a standard ammonium sulfate curve. aC-N and bC-S lyase. aC-N and bC-S lyase activity was measured according to the formation of homocysteine and cysteamine, as detected by gas chromotography/mass spectrometry (GC/MS). Other end products. The procedure described by Weinberg and Holt [23] was used for analysis and identification of amino acid end products. SDS-PAGE Enzyme Kinetics The discontinuous gel system of Laemmli [42] was used for SDS-PAGE analysis. For the determination of Km , Vmax , and kcat , cystalysin was dialyzed into 60 mM potassium phosphate and 0.1 mM EDTA (pH, 7.4). Kinetics were assessed at room temperature in 11 mM potassium phosphate, 170 mM NaCl, and 0.1 mM EDTA (pH, 7.4). All samples were made into 4-mM concentrations in cysteine immediately prior to color development for sulfide quantification, either by addition of cysteine or by dilution of the sample. Km and Vmax were determined by nonlinear leastsquares regression to the Michaelis-Menten equation. The kcat was calculated on the basis of the enzyme concentration estimated from the absorbance at 280 nm, with use of an extinction Antibody Production and Western Blotting (Immunoblotting) Antibody against the purified recombinant cystalysin was produced in New Zealand white rabbits (200 mg of purified protein mixed with Freund’s incomplete adjuvant per injection) as described previously [32]. Western immunoblotting was carried out as described previously [23]. / 9c63$$mr30 02-24-99 10:52:23 cida UC: CID 444 Chu et al. coefficient of 72 mM01 cm01. Inhibition of cystalysin activity was evaluated by varying the concentrations of cysteine and each of the inhibitors. The data were evaluated with use of the equation for the determination of competitive inhibition: V Å Vmax([S]/{[S] / Km(1 / [I]/KI)} CID 1999;28 (March) response of HeO and HeA activity (figure 2, insert). While 36 mM cysteine resulted in significantly increased HeO and HeA activity, this concentration was found to be somewhat toxic to other biological reactions studied. An 18-mM concentration of cysteine was found to give maximum activity with no toxic effects (see below for effect of cysteine on purified cystalysin). by nonlinear least squares. Purification and Characterization of Cystalysin from T. denticola and E. coli Recombinant LC-67 HeO Activity Assay HeO activity was determined with use of sheep RBCs as described by Leady and Smith [46] and as modified by Chu and Holt [31]. HeA Activity Assay HeA activity was determined as described by Chu and Holt [31]. Effect of Potential Compounds and Treatments on the Cysteine Desulfhydrase, HeO, and HeA Activities of the 46-kDa Cystalysin A variety of chemical reagents were evaluated for their effects on H2S-forming activity of the protein. The chemicals were incubated with cystalysin (1 mg/mL) in PBS buffer at 377C for 30 minutes and then mixed with 0.5 mM cysteine for another hour under the same conditions. The effects of pH and temperature on enzyme activity and end-product formation were determined at pH values between 5 and 10 and at temperatures between 07C and 1007C. The production of H2S was determined as described above. For the analysis of effects of proteinase K treatment of cystalysin on end-product formation and on HeO and HeA activities, cystalysin in PBS buffer was incubated with 0.2 mg or 2 mg of proteinase K at 377C for 30 minutes. The reaction products were then analyzed by SDSPAGE and for HeO and HeA activity. Results Growth, Hemoxidation, and Hemolytic Activity of T. denticola As seen in figure 1, growth of T. denticola in standard GM1 medium plus 18 mM cysteine resulted in maximum growth yield after Ç4 days. Hemoxidation (figure 1A; hemoglobinFe/2 to hemoglobinFe/3 and/or sulfhemoglobin) occurred very rapidly, with maximum oxidation occurring between 12 and 24 hours. In comparison, maximum hemolysis (figure 1B) occurred only after Ç2.5 – 3 days. Cysteine also had a significant effect on final growth yield, with maximum yield occurring in a dose-dependent fashion (figure 2). Cysteine was found to be an important stimulator of HeO and HeA activity. In the absence of cysteine, there were very low levels of HeO and HeA activity, while addition of cysteine resulted in a dose-related / 9c63$$mr30 02-24-99 10:52:23 All positive E. coli recombinants overexpressed a protein of 46 kDa [33] and showed H2S-forming activity. One of these (LC-67) was used in the purification of cystalysin from E. coli. Use of DEAE-Sephacel and preparative PAGE electrophoresis resulted in Ç27-fold purification of the protein, with a yield of 42% of the total enzyme activity. Concomitant SDS-PAGE of the various purification steps is shown in figure 3. The soluble fraction from whole cells (figure 3, lane 2) contained a complex polypeptide banding pattern with relative molecular weights ranging from 1.2 kDa to 200 kDa. Ammonium sulfate fractionation (70% – 90% saturation) resulted in substantial purification (figure 3, lane 3). Anion-exchange chromatography on DEAE-Sephacel purified the cystalysin to near homogeneity (figure 3, lane 4). Preparative PAGE (figure 3, lane 5) resulted in the purification of a polypeptide that migrated at a rate identical to that of the native protein (in figure 3, compare lane 5 with lane 6, the latter prepared from T. denticola). Biologically and functionally, the N-terminal amino acid sequence, molecular weight (kDa), amino acid composition, antigenicity, and HeO, HeA, and H2S formation activities of E. coli LC-67 recombinant protein were identical to those of the purified T. denticola cystalysin (data not shown). Characteristics of HeA and HeO Activity of the Purified Recombinant Cystalysin Exposure of RBCs to purified cystalysin resulted in slow but consistent hemoxidation and hemolysis of the RBCs. The activities observed occurred in a dose-dependent fashion, with HeO activity always preceding HeA activity by at least 12 hours. In the presence of cysteine (0.6 mM), there was a 30to 60-fold increase in HeO and HeA activity, which occurred at least 10 – 20 times faster than that seen in the absence of cysteine (data not shown). The HeO and HeA activity of the purified cystalysin appeared to be regulated by various sulfhydryl-containing compounds (figure 4). b-mercaptoethanol (0.5 mM) had a significant enhancing effect on the cysteine (0.6 mM) – dependent HeO and HeA activity of the cystalysin (figure 4). Dithiothreitol (DTT; 0.5 mM), on the other hand, completely inhibited HeO and HeA activity of the cystalysin in the presence of cysteine (figure 4). In the absence of cysteine, cystalysin interacting with b-mercaptoethanol and DTT had no effect on HeO and HeA activity in this experimental condition (data not cida UC: CID CID 1999;28 (March) Cysteine Desulfhydrase from T. denticola 445 Figure 1. Growth (optical density, 660 nm) and hemoxidative (HeO) activity (A) and hemolytic (HeA) activity (B) of Treponema denticola growing in GM-1 medium containing 18 mM L-cysteine (j Å bacterial growth; l Å HeO and HeA activity with use of whole-cell lysates; w Å HeO and HeA activity from ú10 kDa fraction in spent growth supernatant). shown). Homocysteine and methionine also did not alter the HeO and HeA activity of cystalysin. Enzymatic Characteristics of the Cystalysin In addition to exhibiting HeO and HeA functions, cystalysin also exhibited enzymatic activities associated with L-cysteine desulfhydrases (table 1). Incubation of cystalysin with cysteine or cystine resulted in the formation of three end products: H2S, NH3 , and pyruvate. Homocysteine, methionine, oxidized glutathione, b-mercaptoethanol, and dithiothreitol, as well as alanine and serine, were not substrates for cystalysin under the experimental conditions tested (table 1). Incubation of cysta- Figure 2. Effect of L-cysteine on the growth (optical density, 660 nm) of Treponema denticola. A, 0 mM (j), 2 mM (l), 6 mM (w), 18 mM (h), and 36 mM (L) L-cysteine concentrations were added to the basic GM-1 medium (without the L-cysteine routinely present in GM-1 medium); B, the effect of L-cysteine on hemoxidative (HeO; white bar) and hemolytic (HeA; black bar) activities of whole cells of T. denticola (1 1 1010/mL) is shown. / 9c63$$mr30 02-24-99 10:52:23 cida UC: CID 446 Chu et al. CID 1999;28 (March) mocysteine, and cysteamine, instead of H2S. No HeO and HeA activities were observed from the combination of cystalysin plus either of the compounds (table 1). Cystalysin obeys Michaelis-Menten kinetics, with cysteine as the substrate (figure 5). The Km was determined to be 3.4 mM, and the Vmax of 2.52 corresponds to a kcat of 12 sec01 at room temperature. This rate of catalysis supports the idea that cystalysin is a very reactive protein in the conversion of cysteine to its end products. The effect of pH and temperature on enzyme activity were also tested. Maximum H2S production occurred between pH values of 7.8 and 8.0. The enzyme was also sensitive to heat, with temperatures ú507C significantly decreasing enzymatic activity and a temperature of 707C for 30 minutes completely inactivating the enzyme (data not shown). The effects of proteinase K on the integrity and activity of cystalysin were also studied. While proteinase K degrades a large number of different proteins, resulting in both degradation of the protein and concomitant loss of functional activity, this effect was not observed for cystalysin. Treatment of cystalysin with 10 mg or 100 mg of proteinase K per mL cleaved the protein to peptides that were not visualized by SDS-PAGE (data not shown) but still retained HeO and HeA activities and produced H2S (data not shown). Preliminary results indicate that the enzyme activity of the proteinase K digest was localized in a õ10-kDa fraction. Identical results were obtained with pronase at the protease concentrations tested (data not shown). Inhibitor/Activator Studies Figure 3. SDS-PAGE analysis of the purification of the T. denticola 46-kDa cystalysin from the E. coli recombinant strain LC-67. Lane 1, standard-molecular-weight proteins; lane 2, E. coli recombinant LC-67 (soluble fraction); lane 3, 70% – 90% (NH4)2SO4 (ASF) proteins; lane 4, DEAEF proteins (see text); lane 5, final purified recombinant 46-kDa protein from E. coli LC-67 after preparative electrophoresis; lane 6, purified 46-kDa cystalysin from T. denticola TD-4. One hundred milligrams of protein was loaded onto lane 2; 10 mg was loaded onto each of the other lanes. The arrow indicates the location of 46-kDa cystalysin. lysin with reduced glutathione resulted in the formation of small amounts of H2S, while similar incubation with cystathionine and S-b-aminoethyl-L-cysteine (AEC) produced no detectable H2S. However, ammonia and pyruvate were also the end products of cystathionine and AEC. GC/MS analysis of the interaction of cystathionine and AEC with the cystalysin revealed homocysteine and cysteamine as the respective major end products (table 1). Note that cystathionine and AEC also are the substrates of cystalysin but also produced NH3 and pyruvate, ho- / 9c63$$mr30 02-24-99 10:52:23 The effects of potential chemical and physical agents on the interaction of cystalysin with cysteine were tested. Except for b-mercaptoethanol and dithiothreitol, all of the other chemicals, which were selected on the basis of previously reported effects on proteases and other enzymes (CaCl2 , MgCl2 , ZnCl2 , TLCK, PMSF, and benzamidine), had no effect on enzyme activity (data not shown). Use of b-mercaptoethanol and dithiothreitol resulted in substantial increases (fourfold and twofold, respectively) in H2S production, compared with the H2S produced by cysteine alone. a-Amino acids may be expected to inhibit cystalysin, because of their structural similarity to cysteine. Small amino acids (i.e., glycine, serine, and alanine) were found to be competitive inhibitors of cystalysin. The KI values for glycine, Lserine, and L-alanine were 7.4, 15, and 22 mM, respectively (data not shown). The L-amino acids with side chains larger than cysteine were less effective as inhibitors. L-Methionine, L-asparagine, L-valine, and L-threonine, for example, each had KI values ú 150 mM. Discussion We have isolated, purified, and characterized the 46-kDa protein cystalysin with hemolytic activity, from T. denticola cida UC: CID CID 1999;28 (March) Cysteine Desulfhydrase from T. denticola 447 Figure 4. Effect of dithiothreitol (DTT) or b-mercaptoethanol (2ME) on hemoxidative (HeO) activity (A) and hemolytic activity (B) of the cystalysin (2 mg/mL), in the presence of (j) 0.6 mM cysteine and 1% (vol/vol) RBCs; 0.5 mM 2-ME (l) or 0.5 mM DTT (w) was added to the identical assay mixtures. TD-4 and the E. coli recombinant LC-67. Chu and Holt [31] have postulated that the cell-associated cystalysin interacts with RBCs, resulting in oxidation of the hemoglobin iron atom (Fe/2 to Fe/3) to methemoglobin and sulfhemoglobin. 125I-radioautographic studies have localized cystalysin in the RBC plasma membrane, where it results in the formation of irregular holes [30]. These ultrastructural changes are similar to the echinocytes that form upon sulfhemoglobin or choleglobin accumula- Table 1. Substrate specificity of cystalysin. Enzyme product Substrate H2S NH3 Pyruvate Cysteine Homocysteine Cystine AEC [s-(b-aminoethyl)cysteine] Cystathionine Glutathione (reduced) Glutathione (oxidized) Methionine Alanine Serine 2-b-Mercaptoethanol Dithiothreitol / 0 / 0 0 0 0 0 0 0 0 0 / 0 / / / 0 0 0 0 0 0 0 / 0 / / / 0 0 0 0 0 0 0 Other HeO/ HeA activity 0 0 ND Cysteamine Homocysteine 0 0 0 0 0 0 0 FF 0 FFF 0 0 F 0 0 0 0 0 0 NOTE. HeA Å hemolytic; HeO Å hemoxidative; ND Å not done; / Å product detected; 0 Å no product detected in the experimental conditions; F Å enhancement effect of the HeO and HeA activity of the 46-kDa cystalysin (the number of arrows indicates the relative extent of enhancement). / 9c63$$mr30 02-24-99 10:52:23 tion [47]. The formation of the echinocyte appears to be linked to the ability of these altered forms of hemoglobin to extract phospholipids from the inner leaflet of the RBC membrane. Cystalysin’s preferred substrates are sulfur-containing compounds in which the sulfur group is adjacent to both a bmethylene group and an a-amino group (figure 6 and discussion below). As seen in figure 6, cystalysin interaction with cysteine and cystathionine produces stochiometric amounts of NH3 , H2S, and pyruvate, while interaction with (S)-2-aminoethyl-Lcysteine degrades the sulfhydryl-methylene bond to produce cysteamine, NH3 , and pyruvate. Cysteamine cannot be further degraded by cystalysin because of its chemical configuration. In the presence of cysteine, for example, cystalysin interacts with the RBC plasma membrane, where it intercalates, resulting in an apparently rapid rearrangement and degradation of membrane-bound spectrin (data not shown). The formation of the cystalysin channels in the RBC membrane also results in the oxidation of the hemoglobin molecule and the end products of cysteine metabolism, NH3 , H2S, and pyruvate. We conclude, therefore, that cystalysin is a cysteine desulfhydrase (EC 4.4.1.1) [48, 49], and we propose that, as seen in figure 6, cystalysin functions as both an energy source and a source of nitrogen and sulfur for protein synthesis. Although it might be hypothesized that the end products of cystalysin activity may also be involved in the destruction of RBCs, our observations have revealed that even at high concentrations these end products caused negligible HeO and HeA activity (data not shown). The actual mechanism by which the protein participates in RBC oxidation and lysis is still unclear. cida UC: CID 448 Chu et al. Figure 5. Michaelis-Menten kinetics for the production of H2S from cystalysin with L-cysteine as substrate (A). Cystalysin (0.36 mg/ mL) and various concentrations of L-cysteine were incubated at 227C for 40 minutes. A double-reciprocal plot of the enzyme kinetics is shown (B). The curves produced correspond to Vmax Å 2.52 and Km Å 3.4 mM, as determined by a nonlinear least-squares analysis of the upper plot. We propose that in whole cells of T. denticola, intracellular levels of cystalysin are upregulated in the presence of limiting levels or depletion [30] or in the presence of selected sulfhydryl compounds (e.g., cysteine). At least during active periodontal activity, large numbers of RBCs transit into the gingival pocket, / 9c63$$mr30 02-24-99 10:52:23 CID 1999;28 (March) and T. denticola (through hemagglutination [14] or unknown mechanisms) interacts with the RBC plasma membrane, apparently releasing cystalysin into the interior of the RBC, where it interacts with and oxidizes the hemoglobin molecule. Cystalysin has significant protein-sequence homology to the pyridoxal-phosphate-dependent (PPD) aminotransferase family of proteins. The two most studied from this family of enzymes are the Mal Y protein from E. coli [33, 50] (identity of 29%) and C-S lyase from Corynebacterium glutamicum [33, 37] (identity of 29%). The Mal Y protein and C-S lyase have been identified as bC-S lyases, the end products of which are NH3 , pyruvate, and S-containing compounds from amino acids containing a bC-S linkage [37, 38]. Cystalysin exhibited cysteine desulfhydrase activity (deamination). In contrast, cystalysin failed to catalyze gC-S-elimination reactions and also failed to react with aC-N linkages of amino acids without a bC-S linkage (e.g., serine and alanine). However, the enzyme was readily inhibited by small amino acids (glycine, serine, and alanine). The Km toward cysteine (3.6 mM) indicates that the amino acid binds to the active site with a Kd tighter than 3.6 mM. Thus, cysteine is bound more tightly than other, smaller amino acids (glycine and alanine) and the isosteric L-serine or those amino acids with larger side chains. Combined with the substrate profile, our results indicate that the cysteine desulfhydrase activity is the most probable in vivo enzymatic activity of cystalysin. Cystalysin also possesses cystathionase (bC-S lyase) activity, and the proposed chemical reactions of this enzyme with cysteine, cystathionine, and AEC are summarized in figure 6. Cystalysin could also supply NH3 and pyruvate from cysteine-containing compounds, as well as Fe from destruction of RBCs, to other members of the subgingival microbiota for growth and metabolism. Chu and Holt (unpublished data) have observed that both NH3 and pyruvate significantly increased cell yields and produced a shorter generation time in cultures of T. denticola. Cystalysin is not a typical cysteine-dependent hemolysin [31], and the mechanism of hemoxidation and hemolysis may be atypical and complex. It is interesting that b-mercaptoethanol and dithiothreitol (DTT) have opposite effects on the HeO and HeA activities of cystalysin, even though each of the compounds increased H2S production (fourfold and twofold, respectively). The mechanism of DTT inhibition of HeO and HeA activities is not clear. However, since DTT also expressed enhancement of HeO activity when RBCs were initially lysed by sonication, it is more than likely that DTT acts at the level of the membrane to protect the RBCs from attack by either cystalysin or enzyme products. Since cystalysin alone and cysteine as substrate (as control) did not show any HeO and HeA activities within 10 hours, and since cystalysin plus cystathionine or AEC as a substrate (producing NH3 and pyruvate, but without H2S production) did not have HeO and HeA activities (table 1), whether H2S as one of the enzyme products plays an important role in the hemoxidation and hemolysis is to be determined. cida UC: CID CID 1999;28 (March) Cysteine Desulfhydrase from T. denticola 449 Figure 6. Postulated enzymatic action of the Treponema denticola TD-4 cystalysin on cysteine, cystathionine, and (S)-2-aminoethyl-Lcysteine. Arrows indicate the sites of elimination at aC-N and bC-S bonds of the substrates. The end products formed are indicated at the right side of the reactions. The cystalysin from T. denticola may also play a toxic role in the destruction of other host cells [51], including other hemopoietic cells such as lymphocytes, macrophages, and neutrophils [51 – 53]. In this study, cystalysin was shown to be a cysteine desulfhydrase that can function as an iron-regulated metabolic enzyme [30]. High concentrations of H2S in deep periodontal pockets have been reported [54 – 57]. Cystalysin yields significant amounts of H2S as an end product, concomitant with NH3 and pyruvate, reflecting an apparently normal metabolic process for the survival of this microorganism in the host. Since it has been demonstrated that H2S activities are toxic for host cells [28, 29], the cystalysin may also function as a virulence determinant within the confines of the periodontal environment. Acknowledgments The authors are grateful to Dr. Susan Weintraub for her scientific input and participation in the GC/MS analyses and thank Peggy Rifleman for her assistance with the amino acid analyses. Dr. David Kolodrubetz supplied excellent discussion and criticism of this work. References 1. Listgarten MA. 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