YtjE from Lactococcus lactis IL1403 Is a C-S Lyase with α,γ-Elimination Activity toward Methionine

Bacterial strains and culture conditions. E. coli strains DH5α and BL21λ(DE3) (Novagen, Nottingham, United Kingdom), used for cloning and expression of recombinant protein, respectively, were grown aerobically at 37°C in Luria-Bertani (LB) medium. Plasmids were electroporated into E. coli (15), and transformants were selected on LB agar (1.5%) plates supplemented with kanamycin (30 μg ml⁻¹) (Sigma, Dorset, United Kingdom) to select for recombinant pET28b plasmid (Invitrogen, Paisley, United Kingdom). L. lactis IL1403 (11) and L. lactis NCDO 763 (33) were grown at 30°C without agitation in M17 medium supplemented with 0.5% (wt/vol) glucose (G-M17). Growth of bacterial cultures was routinely monitored by measuring the optical density at 600 nm.

Purification of recombinant YtjE protein.Molecular cloning techniques were performed essentially as described by Sambrook et al. (24). To overexpress His-tagged YtjE, a 1.1-kb DNA fragment carrying the L. lactis IL1403 ytjE gene (GenBank accession number AE006422) was amplified from genomic DNA by PCR using primers YTJEF (5′-CCCATGGGAACAAAATATGATTTTACAACCATTCC-3′) and YTJER (5′-TTGCGGCCGCTTAAATTTTGAATGTTTTACGAGTCG-3′), digested with the NcoI and NotI restriction enzymes, and cloned into pET28b (Novagen) to generate pET28b-ytjE. Transformants containing pET28b-ytjE were identified by colony PCR and restriction enzyme analysis of prepared plasmid DNA. Sequencing confirmed that the YtjE coding sequence was in frame with a carboxyl-terminal His₆ tag encoded by the vector. The pET28b-ytjE plasmid was transformed into E. coli BL21λ(DE3), and expression was induced with isopropyl-β-d-thiogalactopyranoside (IPTG). Protein was extracted, and expression of recombinant YtjE was confirmed using the InVision His tag in-gel stain system (Invitrogen). To optimize YtjE production, cultures were grown at 30°C to an optical density at 600 nm of ∼0.5 before we added IPTG (1 mM), and then the cultures were incubated for 3 h at 30°C. Protein was purified using Ni²⁺ affinity chromatography columns (QIAGEN Ltd., Crawley, United Kingdom). Eluted YtjE was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and selected fractions were pooled, dialyzed against a phosphate-buffered saline (pH 7.0) solution containing 10% glycerol, and quantified using the Bradford assay (9).

Preparation of cell samples for enzymatic assays.YtjE activity was assayed using purified His-tagged YtjE protein and cell extracts (CFEs) prepared from IPTG-induced cultures of the E. coli pET28b-ytjE strain. CFEs prepared from cultures of E. coli BL21λ(DE3) carrying empty vector (IPTG induced) and L. lactis IL1403 (grown to logarithmic phase) were used as controls. To prepare CFEs, E. coli and L. lactis cultures (50 ml) were harvested by centrifugation (3,220 × g, 15 min) at 4°C. The cell pellets were washed twice in equal volumes of ice-cold potassium phosphate buffer (50 mM; pH 7.0) and resuspended in 2 ml of ice-cold cell suspension buffer (50 mM potassium phosphate, 10 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 100 μM pyridoxal-5′-phosphate [PLP], pH 7.5). A 1-ml aliquot was then transferred to a microcentrifuge tube containing 1 g of sterile glass beads (1-mm diameter) (Sigma) and vortexed (four times for 2 min, with 1-min intervals on ice). The insoluble fraction and beads were removed by centrifugation (10,000 × g) for 10 min at 4°C, and the supernatant fraction containing soluble protein was aliquoted, frozen in liquid nitrogen, and stored at −80°C until used in enzyme assays.

Aminotransferase activity.Aminotransferase assays were carried out with the R-Biopharm colorimetric l-glutamic acid assay kit (Glasgow, United Kingdom), using different amino acids as substrates (l-methionine, l-isoleucine, l-valine, l-aspartic acid, and l-cysteine). In this assay, the reduced cofactor (NADH) produced by oxidative deamination of l-glutamic acid reacts with iodonitrotetrazolium in the presence of diaphorase to produce a product that absorbs at a wavelength of 492 nm (A₄₉₂). One-milliliter reaction mixtures (70 mM Tris HCl, pH 7.8, 10 mM amino acid, 10 mM α-ketoglutarate, 100 μM PLP, and purified YtjE protein or CFEs) were incubated at 37°C for 1 h and 24 h, and then the reactions were stopped by adding sulfosalicylic acid (3% [wt/vol] final concentration). The colorimetric l-glutamic acid assay was carried out using 30 μl of the reaction mixture, and the increase in A₄₉₂ was monitored during incubation at 37°C for 30 min and 3 h using a Bioscreen (Bioscreen C; ThermoLifeSciences, Basingstoke, United Kingdom). A standard curve was obtained using different concentrations of l-glutamic acid. CFEs prepared from L. lactis NCDO 763, a strain previously shown to exhibit aminotransferase activity toward Met, were used as a positive control (33). Met aminotransferase activity of the purified YtjE protein was also assayed by measuring the formation of KMBA from l-methionine by reverse-phase high-pressure liquid chromatography (HPLC), using an Agilent HP1100 HPLC system with a PDA UV-vis detector. Reaction products were separated using a Gemini C₁₈ column (150 mm by 2.0 mm by 5 μm) (Phenomenex, Macclesfield Cheshire, United Kingdom) thermostated at 30°C. The mobile phase was a linear gradient of acetonitrile plus 0.1% trifluoroacetic acid (solvent B) in Milli-Q water (Millipore Co., Bedford, MA) plus 0.1% trifluoroacetic acid (solvent A) at a flow rate of 0.3 ml min⁻¹. The sample injection volume was 20 μl. The elution profile was monitored at 220 nm, and peaks were identified by comparing retention times with standards.

C-S lyase activity.C-S lyase activity of YtjE was determined by measuring the formation of (i) free thiol groups, (ii) keto acid, and (iii) ammonia, using l-cysteine, l-cystathionine, l-cystine, or l-methionine as the substrate.

The formation of free thiol groups was determined according to a method previously described by Uren (30). Reaction mixtures containing 0.2 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 100 μM PLP, substrate (at different concentrations), and either YtjE or CFEs suspended in a 100 mM Tris HCl (pH 8.0) solution were incubated at 37°C for 30 min and 2 h, and the increase in A₄₁₂ was measured at 2-min intervals. The molar absorption coefficient value used for aryl mercaptide was 13,200 liter mol⁻¹ cm⁻¹, with 1 enzyme unit representing the formation of 1 μmol aryl mercaptan min⁻¹. The kinetic parameters were computed from the Lineweaver-Burk transformation (V⁻¹ versus S⁻¹) of the Michaelis-Menten equation, where V is the formation rate of free thiol groups (μmol min⁻¹ mg⁻¹ protein) and S is the concentration (mM) of each substrate. To further characterize enzyme activity, the purified YtjE protein was also incubated with different inhibitors (at the indicated concentrations) (Table 1) before the formation of free thiol groups was measured, using l-cystathionine (2 mM) as the substrate.

Keto acid formation was detected as described previously (16). Briefly, the assay was carried out in 1 ml of 100 mM Tris HCl (pH 8.0) containing 100 μM PLP, 2 mM substrate, and various amounts of protein. The reaction was started by adding protein, incubated for 60 min at 37°C, and stopped by adding 0.5 ml of 4.5% trichloroacetic acid. The suspension was centrifuged, and 0.5 ml of the recovered supernatant was added to 0.5 ml of 0.05% 3-methyl-2-benzothiazolinone hydrazone (in 1 M sodium acetate, pH 5.2) and incubated at 50°C for 30 min. The amounts of keto acid produced were spectrophotometrically measured (A₃₃₅). The formation of pyruvate was also assayed in a lactate dehydrogenase-coupled reaction mixture containing 100 mM Tris HCl (pH 8.0), 2 mM substrate, 100 μM PLP, 200 μM NADH, and 10 μg of lactate dehydrogenase. Reaction mixtures were prewarmed at 37°C prior to the addition of YtjE or CFEs, and the reduction in A₃₄₀ due to oxidation of NADH to NAD was then monitored as a measure of the amount of pyruvate produced.

The formation of ammonia was assayed by measuring the amount of NADH oxidized in the presence of α-ketoglutarate and glutamate dehydrogenase. One-milliliter reaction mixtures containing 100 mM Tris HCl (pH 8.0), 2 mM substrate, 100 μM PLP, and either YtjE or CFEs were incubated at 37°C for 1 h and 24 h. The ammonia assay was carried out using 30 μl of the reaction mixture. The oxidation of NADH was monitored by measuring the decrease in absorbance at 340 nm at intervals of 3 min for 1 h.

Detection of C-S lyase activity by in situ staining. l-Cysteine desulfhydrase activity was confirmed by visualizing enzyme activities (32). YtjE or CFEs from E. coli and L. lactis were added to 150 μl of a visualizing solution containing 100 mM triethanolamine HCl (pH 7.6), 100 μM PLP, 0.5 mM bismuth trichloride, 10 mM EDTA, 1% Triton X-100, and 5 mM l-cysteine. These mixtures were then incubated at 37°C to detect the development of black precipitate formed by the reaction of sulfide with bismuth. C-S lyase activities (toward l-cysteine) of YtjE and CFEs were also monitored using Tris-glycine gels (Invitrogen) run under nondenaturing conditions. Following electrophoresis (20 mA at 4°C for 2 h), gels were incubated at 37°C in 10 ml of the visualizing solution to detect black precipitate at the position of enzyme activity.

Identification of reaction products of l-cystathionine due to YtjE activity.Amino acid products of YtjE activity toward l-cystathionine were identified by HPLC analysis (at 214 nm) of reaction mixtures containing DTNB derivatives of free thiol group-containing amino acids, as described by Bruinenberg et al. (10).

Determination of cysteine.The specificity of YtjE activity toward l-cystathionine (β/γ) was also assayed by the ninhydrin procedure (18). The formation of cysteine due to YtjE cystathionine γ-lyase activity was determined based on the specific reaction of cysteine with acid nynhydrin reagent (250 mg of ninhydrin in a mixture of 6 ml of acetic acid and 4 ml of HCl) by measuring the increase in absorbance (A₅₆₀).

VSCs produced by YtjE.YtjE activity toward l-methionine and l-cysteine was also determined by analyzing the production of VSCs. Reactions were carried out in 100 mM Tris HCl (pH 8.0) containing 100 μM PLP and either l-methionine or l-cysteine (5 mM) mixed with YtjE protein or CFEs and incubated at 25°C for 48 h, with samples taken at different time intervals. The products formed were determined by solid-phase microextraction and gas chromatography-mass spectrometry (GC-MS). Samples were allowed to warm to room temperature before the headspace was sampled for 15 min with an 85-μm Carboxen-polydimethylsiloxane fiber (Supelco, Dorset, United Kingdom). The fibers were cleaned by heating at 250°C before use and after injection. Volatile compounds were analyzed by a gas chromatograph (model 6890; Hewlett Packard) connected to an HP 5973 mass selective detector. Samples were injected manually onto a DB-FFAP column (30 m by 0.32 mm; 1-μm film thickness) (Agilent J&W GC; Agilent, Cheshire, United Kingdom) at a helium flow rate of 2 ml min⁻¹ for analysis. The oven temperature was held at 40°C for 5 min, then programmed to increase to 100°C at a rate of 5°C min⁻¹, and held at this temperature for 1 min before increasing again to 240°C at a rate of 30°C min⁻¹; the temperature of the column was then held at 240°C for 1 min before returning to 40°C. The injector port was maintained at a temperature of 250°C and operated in the splitless mode. The MS source temperature was 200°C, and the transfer line was 250°C. The mass selective detector was scanned from 30 to 300 m/z at a speed of 0.72 scans s⁻¹. Areas of peaks were calculated using ChemStation software (Agilent Technologies UK Ltd., United Kingdom), selecting the beginning and end of molecular peaks manually. Sulfur-containing compounds were identified by comparing retention times and mass spectra with those obtained using standards prepared individually or as mixtures by using pure stocks of DMDS and DMTS (Sigma).

Expression and purification of L. lactis IL1403 YtjE.To obtain purified protein for enzymatic analysis, the ytjE gene from L. lactis IL1403 was cloned and overexpressed in E. coli as a C-terminal His-tagged fusion protein. The cloned ytjE gene was identical to the native gene, except for the introduction of a triplet of bases (GGA) resulting in the addition of an extra amino acid (Gly) at position 2 at the N terminus of the recombinant YtjE protein. SDS-PAGE analysis and His-tag in-gel staining of IPTG-induced CFE of the recombinant E. coli strain confirmed expression of YtjE, which was detected in both the soluble and insoluble fractions, the latter probably due to the formation of insoluble aggregates as a result of the high level of expression (Fig. 1). His-tagged YtjE protein was then purified under native conditions by affinity chromatography on nickel-nitrilotriacetic acid resin, and its purity was confirmed by SDS-PAGE (Fig. 1). SDS-PAGE analysis of the recombinant protein showed a band of approximately 55 kDa, a molecular mass higher than that predicted using its amino acid sequence (46 kDa).

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FIG. 1.

SDS-PAGE analysis of CFEs prepared from an IPTG-induced culture of E. coli pET28-ytjE (lanes: 4, total fraction; 5, soluble fraction; 6, insoluble fraction). CFEs prepared from an IPTG-induced culture of E. coli carrying empty vector (lanes: 1, total fraction; 2, soluble fraction; 3, insoluble fraction) were included as a control. Purified His-tagged YtjE protein is also shown (arrow; lane 7). The molecular mass markers are also indicated (kDa).

Methionine aminotransferase activity of YtjE.In the genome of L. lactis IL1403, the product of ytjE has been suggested to be an aminotransferase specific for Met (6). The putative Met aminotransferase activity of YtjE was therefore assayed by measuring the formation of l-glutamic acid, using Met as the substrate and α-ketoglutarate as an acceptor of the amino group. Similar levels of l-glutamic acid (mean ± standard deviation) were detected in reaction mixtures containing CFEs from the E. coli strains expressing (58 ± 9 nmol min⁻¹ mg⁻¹ protein) or not expressing (51 ± 11 nmol min⁻¹ mg⁻¹ protein) recombinant YtjE, and no l-glutamic acid could be detected with the purified protein. As expected, high levels of glutamate were obtained when we used CFE from L. lactis NCDO 763 (112 ± 10 nmol min⁻¹ mg⁻¹ protein). When tested, purified YtjE was also shown not to exhibit aminotransferase activity toward l-valine, l-isoleucine, l-phenylalanine, l-cysteine, and l-aspartic acid. To confirm these results, the putative Met aminotransferase activity was also assayed by reverse-phase HPLC analyses as described in Materials and Methods. KMBA, a product of Met transamination, could not be detected. These results suggest that YtjE is not a Met aminotransferase.

C-S lyase activity.An interrogation of the database, using the deduced YtjE amino acid sequence, revealed relatively high levels of homology to the Lcd protein (59% identity) of Streptococcus anginosus, the PatB protein (38%) of Bacillus subtilis, the PatC protein (33%) of Lactobacillus delbrueckii, and the MalY protein (28%) of E. coli (Fig. 2). These proteins have previously been described as C-S lyases with α,β-elimination activity (4, 5, 32, 34). On the basis of this information, the activity of YtjE (in CFE and as purified protein) was investigated by assessing its ability to generate free thiol groups, α-keto acid, and ammonia, using the sulfur-containing compounds l-cysteine, l-cystathionine, l-cystine, and l-methionine as substrates. When their CFEs were compared, the degradation of these substrates measured by the formation of free thiol groups (reacting with DTNB) was higher in the E. coli pET28b-ytjE strain than in the vector control, thereby suggesting a role for YtjE. This was confirmed using purified YtjE protein, which, under these study conditions, exhibited higher activity toward l-cystine (2,230 ± 120 mU mg⁻¹ protein) than toward l-cystathionine (650 ± 10 mU mg⁻¹) and l-methionine (40 ± 1 mU mg⁻¹). The apparent K_m values determined from Lineweaver-Burk plots for l-cystine, l-cystathionine, and l-methionine were 0.46 ± 0.10 mM, 0.27 ± 0.05 mM, and 0.87 ± 0.18 mM, respectively. The relative activities of YtjE toward these substrates were replicated when enzyme activity was determined by measuring the formation of keto acids and ammonia. Furthermore, enzymatic activity toward these substrates always resulted in the formation of a free thiol group, a keto acid component, and ammonia as reaction products, thus confirming the C-S lyase activity of YtjE.

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FIG. 2.

Alignment of YtjE amino acid sequence from L. lactis IL1403 with Lcd from S. anginosus, PatB from B. subtilis, PatC from L. delbrueckii, and MalY from E. coli. Boxes indicate identical amino acid residues in all the sequences analyzed. The four residues that are invariant in all aminotransferases (▾) (1) are shown. The Lys₂₃₃ residue is the potential binding site of PLP. Residues typical for aminotransferases (•) and those typical for trans-sulfuration enzymes (○) are also indicated.

C-S lyase activity toward l-cysteine, also known as cysteine desulfhydrase activity, was assayed by in situ staining. Using this approach, cysteine desulfhydrase activity was conveniently monitored in nondenaturing gels by using l-cysteine as the substrate. Hydrogen sulfide (H₂S) reacts with bismuth to produce an insoluble product which forms brown-to-black bands on the gels (32). Electrophoresis under nondenaturing conditions of purified YtjE revealed a single dark-brown band (Fig. 3), confirming the ability of the YtjE protein to catalyze the α,β-elimination reaction of l-cysteine to H₂S. The same band was also detected in CFEs from a recombinant E. coli strain expressing YtjE and L. lactis IL1403.

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FIG. 3.

Cysteine desulfhydrase activity by in situ staining. YtjE activity was monitored in Tris-glycine gels under nondenaturing conditions, using l-cysteine as the substrate. Lanes: 1, 5 μg purified recombinant protein; 2, 2.5 μg purified recombinant protein; 3, CFE from E. coli recombinant strain expressing YtjE; 4, CFE from E. coli carrying empty vector; 5, CFE from L. lactis IL1403.

It has been reported that l-cystathionine degradation can lead to the production of different free thiol-containing compounds, homocysteine and cysteine, via two reactions involving α,β-elimination and α,γ-elimination activities, respectively. When tested, homocysteine was identified to be the sole free thiol-containing compound of l-cystathionine degradation due to the purified YtjE protein by HPLC analysis of DTNB-derivatized reaction products (data not shown). Moreover, the formation of cysteine could not be detected using the ninhydrin method. These results strongly suggest therefore that the YtjE enzyme exhibits only α,β-elimination activity toward l-cystathionine.

The effects of selected inhibitors on YtjE activity, measured by the formation of free thiol groups using l-cystathionine as the substrate, are summarized in Table 1. As with most cystathionine β/γ-lyases (2, 10, 25), YtjE activity was strongly inhibited by carbonyl reagents such as hydroxylamine and 3-methyl-2-benzothiazolinone hydrazone, known inhibitors of PLP-dependent enzymes. The chelating reagent EDTA had no inhibitory effect on activity, while the addition of 4% NaCl resulted in a threefold reduction in enzyme activity. Contrary to the aforementioned cystathionine β/γ-lyases, however, YtjE was also sensitive to the thiol-reactive agent iodoacetic acid.

Formation of volatile sulfur compounds.YtjE activity toward l-cysteine and l-methionine was also assayed by GC-MS in order to identify the VSCs formed. The VSCs formed were H₂S (relative peak area of 110,012 ± 10,810 units μg⁻¹ after 24 h of incubation) from l-cysteine and MTL, DMDS, and DMTS from l-methionine (Fig. 4). These compounds could not be detected in control samples incubated in the absence of YtjE. These results indicate that in addition to demonstrating α,β-elimination activity toward l-cysteine, YtjE can also exhibit α,γ-elimination activity with l-methionine as the substrate. As shown in Fig. 4, the formation of MTL increased with incubation time along with the formation of DMDS and DMTS. This is not surprising, as MTL is a highly unstable sulfur compound that quickly reacts to form the oxidized and more stable compound DMDS and, to a lesser extent, DMTS.

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FIG. 4.

Production of MTL, DMDS, and DMTS by coincubating l-methionine with the purified YtjE protein. Values are the means of three independent measurements. The standard errors of the means are also indicated.