Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase

Authors


*Corresponding author. Tel.: +1 (814) 863-5721; Fax: +1 (814) 863-6217, E-mail address: jgf3@psu.edu

Abstract

Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize. Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain. The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M. thermophila and possibly form an operon. When M. thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.

1Introduction

Cysteine plays a critical role in the structure, stability and catalytic function of many proteins in all domains of life. Cysteine is also the major source of sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteria and Eucarya domains. Two routes for cysteine biosynthesis in nature have been documented. Serine transacetylase and O-acetylserine sulfhydrylase catalyze steps in pathway I (Fig. 1, reactions 1–2) [1]. Cystathionine β-synthase and cystathionine γ-lyase catalyze steps of pathway II (Fig. 1, reactions 5–6) [1]. Plants and members of the Bacteria domain synthesize cysteine and fix sulfur via pathway I, and also fix sulfur by synthesizing homocysteine (Fig. 1, reactions 3–4); however, most cannot utilize homocysteine for cysteine biosynthesis. Fungi fix sulfide (Fig. 1, reactions 2 and 4) and can theoretically synthesize cysteine using pathways I and II. Although enzymes for both pathways are present in the yeast Saccharomyces cerevisiae, only pathway II is used for cysteine biosynthesis [2]. On the other hand, the yeasts Schizosaccharomyces pombe[3], Pichia membranofaciens and Candida valida[4,5] lack cystathionine β-synthase and cystathionine γ-lyase, indicating these organisms must synthesize cysteine through pathway I.

Figure 1.

Known cysteine biosynthetic pathways. Enzymes catalyzing the steps are: 1, serine transacetylase; 2, O-acetylserine sulfhydrylase; 3, homoserine transacetylase; 4, homocysteine synthase; 5, cystathionine β-synthase; 6, cystathionine γ-lyase.

Although the genomes of five members of the Archaea have been sequenced to date, the data offer little understanding of cysteine biosynthesis. The Methanococcus jannaschii[6], Methanobacterium thermoautotrophicum[7] and Archaeoglobus fulgidus[8] genomes contain no open reading frames (ORFs) having a deduced sequence with significant identity to enzymes of either pathway for cysteine biosynthesis. The genome of Aeropyrum pernix[9] contains ORFs with deduced sequence similarity to cystathionine β-synthase and cystathionine γ-lyase. The genome of Pyrococcus horikoshii[10] contains an ORF with deduced sequence similarity to cystathionine γ-lyase. However, it is not known whether these genes are expressed or if the gene products have the expected enzyme activities. No ORFs are present in the genomes of P. horikoshii and A. pernix with a deduced sequence having significant identity to enzymes of pathway I for cysteine biosynthesis.

In Methanosarcina barkeri, two genes were identified that had significant sequence similarity to cysK (encoding O-acetylserine sulfhydrylase) and cysE (encoding serine transacetylase) [11]. The cysK gene was found to complement a cysteine auxotrophic Escherichia coli strain, indicating that the gene encodes O-acetylserine sulfhydrylase. However, cysK expression was not demonstrated in M. barkeri and no other data were reported supporting cysK involvement in cysteine biosynthesis. Notably in S. cerevisiae, both pathway I enzymes are present but they have an undefined role distinct from cysteine biosynthesis [12]. However, it was recently shown that O-acetylserine sulfhydrylase is expressed in Methanosarcina thermophila and the enzyme has properties suggesting involvement in cysteine biosynthesis [13]. Here we provide further evidence that pathway I is used for cysteine biosynthesis and pathway II is not present.

2Materials and methods

2.1M. thermophila growth media and cell extract preparation

General anaerobic methods for the growth of M. thermophila and preparation of extracts were as described [13,14]. Minimal media [14] contained the following constituents in demineralized water at the final percent concentrations (w/v): NH4Cl, 0.13; K2HPO4, 0.11; KH2PO4, 0.09; CaCO3, 0.0002; p-aminobenzoic acid, 0.000004; sodium acetate·3H2O, 1.4; Fe(NH4)2(SO4)2·6H2O, 0.001; Na2CO3, 0.25; resazurin, 0.0001; and 1% (v/v) each of vitamin and trace mineral solutions as described [15]. The following were added individually or in combination to supply the sulfur source (final concentration in the media (w/v)): Na2S·9H2O, 0.025; cysteine–HCl·H2O, 0.025.

Cultures were transferred during late-exponential phase 10 successive times into fresh media containing either cysteine or sulfide or both (1 ml into 60 ml) before growth was recorded, to ensure no carryover of cysteine or sulfide from the original inoculum.

2.2S. cerevisiae w303a growth media and cell extract

S. cerevisiae w303a was a gift from Robert J. Durso. YPD medium contained 2% glucose, 2% peptone and 1% yeast extract [16,17]. Growth was at 30°C. Cell homogenates were prepared as described previously [16]. Cells were harvested during late-exponential phase, and washed twice with STE (10 mM Tris–Cl (pH 7.4), 1 mM EDTA, 100 mM NaCl). Washed cells were resuspended 1:1 in WCE (20 mM potassium phosphate (pH 7.2), 100 nM pyridoxal phosphate, 250 nM EDTA, 1 mM PMSF, 1 μg ml−1 pepstatin A, 10 μg ml−1 leupeptin, 350 mM NaCl, 1% Tween 20 and 10% glycerol) and frozen in small droplets in liquid nitrogen until use. Cells were broken by grinding the frozen droplets with a liquid nitrogen-chilled mortar and pestle for 15 min (until a fine powder remained). The cell homogenate was centrifuged twice at 13 000×g for 10 min at 4°C, and the supernatant was stored in 0.05-ml aliquots at −80°C.

2.3Protein assay

Protein concentrations were determined by the method of Bradford [18], using Bio-Rad dye reagents and bovine serum albumin (Pierce) as a standard.

2.4Enzyme assay

O-Acetylserine sulfhydrylase activity was measured as described previously [19]. Cystathionine γ-lyase activity assays were conducted as described previously [20] using the method of Droux [19] to detect the product cysteine. Cystathionine β-synthase activity was measured as described previously [21]. All reactions were performed at 40°C with M. thermophila cell extract alone, and 30°C with S. cerevisiae cell extract alone or together with M. thermophila cell extract. Protein concentrations were 4 mg ml−1 per assay per organism (thus 8 mg ml−1 total protein concentration when using both cell extracts).

2.5Gene isolation

M. thermophila DNA was a gift from Birgit Alber. Primers were designed using the M. barkeri cysK gene [11]. The cysK gene from M. thermophila was amplified through PCR (Ericomp DeltaCycler II™ System of the EasyCycler™ Series) and cloned into a pTAdvantage vector (Clontech). Using primers to the M. thermophila cysK gene, upstream DNA was obtained with the Universal GenomeWalker™ kit (Clontech), and downstream DNA was obtained using M. thermophila DNA digested with EcoRI and ligated into a pUC 21 vector (a gift from Robert Barber). The resultant upstream and downstream DNA were cloned into pSTBlue-1 AccepTor Vector™ (Novagen). All transformants were obtained following the manufacturers’ instructions (Novagen). Plasmids were purified using QIAprep Spin Miniprep Columns (Qiagen), and were sequenced using a Perkin Elmer ABI377 (Penn State University Sequencing Facility).

The BLAST blastx program [22] was used to search the non-redundant sequence databases at the National Center for Biotechnology Information (Bethesda, MD, USA) for genes with a similar sequence to the ORFs upstream (nifS and the hypothetical protein) and downstream (cysE) of cysK. The hypothetical protein from M. thermophila, M. barkeri[11], and AF0184 and AF0566 from A. fulgidus[8] were aligned by using Pattern-Induced (local) Multiple Alignment 1.4 from the Baylor College of Medicine Search Launcher [23].

3Results

3.1Growth of M. thermophila with either cysteine, sulfide or cysteine plus sulfide as the source of sulfur

Growth in minimal media, as determined by gas production, was similar in media that contained either cysteine plus sulfide, only sulfide, or only cysteine as the sole sulfur source (Fig. 2). There was no significant difference in cell yield (lyophilized dry weight) for any of the cultures at the end of 10 days of growth (+Cys/+S2−: 22±4 mg; −Cys/+S2: 24.7±0.9 mg; +Cys/−S2−: 20±5 mg). The results demonstrate that M. thermophila synthesizes cysteine de novo and can use either sulfide or cysteine as the sole sulfur source.

Figure 2.

Growth of M. thermophila in minimal media with cysteine and sulfide ▪, with sulfide only ▴ and with cysteine only • as sulfur source. Cultures (60 ml) were grown in 120-ml serum bottles.

3.2Activities in cell extracts of enzymes catalyzing steps in known cysteine biosynthetic pathways

When M. thermophila was grown in minimal media without cysteine and with sulfide, O-acetylserine sulfhydrylase activity in cell extract was elevated over 50-fold compared to growth in media containing both sulfide and cysteine (Table 1). When grown in minimal media with only cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity in extract was elevated over 150-fold compared to growth in media containing both sulfide and cysteine (Table 1). No other protein fractions with O-acetylserine sulfhydrylase activity were detected during the purification of the O-acetylserine sulfhydrylase from M. thermophila[13], indicating that the enzyme purified is responsible for the elevated activities.

Table 1.  Specific activity of cysteine biosynthetic enzymes in M. thermophila grown in minimal media with different sulfur sources, and S. cerevisiae grown in standard media
  1. amU=nmol cysteine min−1.

  2. bmU=nmol cystathionine min−1.

  3. cLimits due to sensitivity of assay.

  4. dValues in parentheses are with an equal amount of S. cerevisiae extract added, and are calculated based on S. cerevisiae extract alone.

  5. eN/A=not assayed.

Organism and sulfur sourceCystathionine γ-lyase (mU mg−1)aCystathionine β-synthase (mU mg−1)bO-Acetylserine sulfhydrylase (mU mg−1)a
M. thermophila cysteine and sulfide<0.04c (0.2±0.06)d<0.3c (0.7±0.3)0.25±0.05
M. thermophila sulfide<0.04c (0.2±0.05)<0.3c (0.8±0.4)14±1
M. thermophila cysteine<0.04c (0.2±0.02)<0.3c (0.9±0.1)45±4
S. cerevisiae yeast extract and peptone0.3±0.031.2±0.4N/Ae

Cystathionine γ-lyase and cystathionine β-synthase activities were not detected in cell extracts of M. thermophila grown under the conditions examined, including in the absence of cysteine (Table 1). Activities of these enzymes were detected in extracts of S. cerevisiae grown under conditions where both enzymes are expressed for the synthesis of cysteine [16,17]. Both enzyme activities from S. cerevisiae cell extract were nearly fully recovered when added to the individual M. thermophila cell extracts. These results indicate that no enzyme or other component in the cell extracts of M. thermophila significantly masks either cystathionine γ-lyase or cystathionine β-synthase activities.

3.3Organization of genes that encode O-acetylserine sulfhydrylase and serine transacetylase

The cysK gene encoding the O-acetylserine sulfhydrylase from M. thermophila was obtained by PCR amplification of genomic DNA using primers derived from the published sequence for cysK[11]. The deduced sequence of the PCR-amplified DNA was highly identical to O-acetylserine sulfhydrylase sequences from plants and prokaryotes (53% identity with Oryza sativa, 63% identity with Mycobacterium tuberculosis, 89% identity with M. barkeri). The first 20 N-terminal residues deduced from the sequence were 100% identical to those determined for the O-acetylserine sulfhydrylase isolated from M. thermophila[13]. The 1-kb sequence downstream and 4-kb sequence upstream of cysK were obtained by PCR amplification using primers derived from the M. thermophila cysK sequence. Analysis of the flanking sequences identified a partial ORF downstream of cysK (Fig. 3A), the truncated deduced sequence (243 residues) of which was found to have high identity to cysE genes present in plants and prokaryotes (39% identity with Arabidopsis thaliana, 55% identity with Azotobacter vinelandii, 87% identity with M. barkeri). Upstream of cysK, and transcribed in the opposite direction, is a partial ORF designated nifS (Fig. 3A). The deduced sequence of nifS has 50% identity to nifS from A. vinelandii. Situated between the M. thermophila cysK and nifS genes is an ORF encoding a hypothetical protein with 180 residues that is transcribed in the same direction as cysK and cysE (Fig. 3A). M. barkeri has the same gene organization (Fig. 3A). The deduced sequence of the M. barkeri ORF is 76% identical to the M. thermophila hypothetical protein (Fig. 3B). The hypothetical proteins encoded by these ORFs have significant identity to hypothetical proteins in the archaeon A. fulgidus[8] (AF0184 and AF0566 have 34% and 33% identity to the M. thermophila hypothetical protein, respectively) (Fig. 3B). Both A. fulgidus hypothetical proteins are clustered with nifU and nifS homologs that are transcribed in the same direction as the hypothetical proteins.

Figure 3.

Gene arrangement and sequence alignment of the hypothetical protein from M. thermophila and M. barkeri. A: Gene arrangement of cysK, cysE, ORF and nifS in M. thermophila (M.t.) and M. barkeri (M.b.). B: Sequence alignment of the hypothetical protein of M. thermophila, M. barkeri and A. fulgidus (AF0184, AF0566). Identical residues are indicated in boldface. M.t. cysE and M.b. nifS sequences are truncated. The M. thermophila DNA sequence has been deposited at GenBank and the accession number is AF276772.

4Discussion

The genomic sequences of five phylogenetically and metabolically diverse members of the Archaea provide little insight into mechanisms of cysteine biosynthesis in this domain. Although cysK was identified in M. barkeri[11], biochemical evidence for expression of cysK and involvement of CysK in cysteine biosynthesis was not reported. However, definitive biochemical evidence for expression of O-acetylserine sulfhydrylase and characterization of the enzyme was recently reported for M. thermophila[13]. Moreover, the enzyme from M. thermophila exhibits positive co-operativity indicative of involvement in a biosynthetic pathway [13]. Thus, the biochemical results reported for the O-acetylserine sulfhydrylase from M. thermophila suggest that pathway I operates in this archaeon for the synthesis of cysteine. Here we provide physiological and genetic evidence for involvement of this pathway. First, cystathionine γ-lyase and cystathionine β-synthase activities were not detected, indicating the only other pathway known for the synthesis of cysteine was not operable in M. thermophila under the conditions tested. Second, O-acetylserine sulfhydrylase activity in cells grown in media without cysteine and with sulfide was elevated 50-fold compared to the activity in cells grown in the presence of cysteine and sulfide where activity was fully repressed. Third, the cysK gene encoding O-acetylserine sulfhydrylase was found adjacent to an ORF with high identity to genes encoding serine transacetylase (cysE). Both cysK and cysE in M. thermophila are transcribed in the same direction and possibly form an operon.

Our previous results [13] describing the expression and allosteric properties of the O-acetylserine sulfhydrylase from M. thermophila, and the results reported here, suggest that the cysK gene identified in M. barkeri is also involved in cysteine biosynthesis and this pathway may operate in all Methanosarcina species. The apparent absence in the genomes of M. jannaschii and M. thermoautotrophicum of ORFs with identity to genes known to encode O-acetylserine sulfhydrylase and serine transacetylase suggests this pathway does not operate in all methanoarchaea; however, biochemical analyses are necessary to rule out convergently evolved enzymes with the requisite activities. Likewise, biochemical approaches will be necessary to determine the pathways of cysteine biosynthesis in other members of the Archaea.

It was surprising to find that both cysteine and sulfide were necessary to fully repress the expression of O-acetylserine sulfhydrylase activity, assuming the enzyme functions only in the synthesis of cysteine. It was also unexpected to find a nearly 3-fold elevation of activity in cells grown with cysteine compared to cells grown with sulfide as the sole sulfur source. These results indicate another role for O-acetylserine sulfhydrylase other than cysteine biosynthesis. One possible alternative role is that O-acetylserine sulfhydrylase can mobilize sulfur from cysteine for other biosynthetic reactions. Additional observations are consistent with this hypothesis. First, growth with cysteine as the only sulfur source shows that cysteine can be imported, and, therefore, O-acetylserine sulfhydrylase is not needed for cysteine biosynthesis under these conditions. Second, the O-acetylserine sulfhydrylase purified from M. thermophila has a robust desulfurase activity of 240 nmol H2S min−1 mg−1 (unpublished results). This activity compares favorably with the activity (89.4 nmol H2S min−1 mg−1) reported for A. vinelandii NifS [24] which catalyzes the desulfurization of cysteine for sulfide incorporation into iron–sulfur clusters. Third, during the purification of O-acetylserine sulfhydrylase from M. thermophila grown with cysteine as the sole sulfur source [13], only one other enzyme fraction with cysteine desulfurase activity was detected. This additional fraction was responsible for less than 10% of the total activity in extracts, indicating that the O-acetylserine sulfhydrylase is responsible for greater than 90% of the desulfurase activity in cells grown with cysteine as the only source of sulfur. When M. barkeri is grown with cysteine as the sole sulfur source, cysteine is rapidly taken up by the cells and desulfurated (as noted by the simultaneous evolution of sulfide into the media during cysteine uptake) [25]. If, as in M. thermophila, greater than 90% of the desulfurase activity is catalyzed by O-acetylserine sulfhydrylase then it is likely that this enzyme is largely responsible for the release of sulfide from cysteine in M. barkeri. Finally, the E. coliO-acetylserine sulfhydrylase also has desulfurase activity, and has been shown to aid in iron–sulfur cluster formation [26] similar to NifS.

A nifS gene is upstream of cysK and cysE in both M. thermophila and M. barkeri. NifS mobilizes sulfur from cysteine for the formation of Fe–S clusters [24]. Though it was originally discovered as one of a dozen enzymes required for nitrogen fixation in A. vinelandii, nifS has been found in non-nitrogen fixing organisms, suggesting a universal role for NifS in Fe–S cluster formation. It has been proposed that NifU may function in cluster formation by sequestering inorganic Fe, aiding in the release of sulfide from NifS [27], or donating Fe2S2 units for the synthesis of Fe–S clusters [28]. The hypothetical proteins encoded by the ORFs in M. barkeri and M. thermophila between nifS and cysK are similar to hypothetical proteins encoded by ORFs clustered with nifU and nifS homologs in the archaeon A. fulgidus. The location of these ORFs in archaeal species suggests the possibility that the encoded hypothetical proteins may play a role in conjunction with CysK or NifS, possibly to assist in the mobilization of sulfur from cysteine or Fe–S cluster synthesis as proposed for NifU.

Acknowledgments

This work was funded by NASA Ames Cooperative Agreement NCC21059 and DOE Grant DE-FG02-95ER20198. We thank Jennifer Loveland-Curtze for help with cell lyophilization, Robert J. Durso for donating the S. cerevisiae strain and helping with culturing and making cell extract. We also thank Birgit Alber for the M. thermophila DNA, Robert Barber for the plasmid DNA library, and Jonna Coombs, Peter P. Sheridan and Robert Barber for critical reading of the manuscript.

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