Biosynthesis of a sulfonolipid in gliding bacteria

Gliding bacteria of the genus Cytophaga synthesize sulfonolipids (1,2) that contain capnine (1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Studies of the incorporation of radiolabeled compounds by C. johnsonae show that cysteate is utilized preferentially to both cystine and inorganic sulfate as a precursor of capnine sulfur and to both cystine and serine as a precursor of carbons 1 and 2 of capnine. The results are consistent with a pathway in which capnine is formed by condensation of cysteate with a fatty acyl CoA. Cystine, added as the sole sulfur source in the presence of glucose, provides the sulfur but not the carbon for capnine. Hence, these cells form cysteate not by direct oxidation of cystine (or cysteine), but by transfer of its sulfur to a different carbon compound.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Transport of Cysteate by Synaptosomes Isolated from Rat Brain: Evidence that It Utilizes the Same Transporter as Aspartate, Glutamate, and Cysteine Sulfinate

Synaptosomes isolated from rat brain accumulated cysteic acid by a high-affinity transport system (Km = 12.3 +/- 2.1 microM; Vmax = 2.5 nmol mg protein-1 min-1). This uptake was competitively inhibited by aspartate (Ki = 13.3 +/- 1.8 microM) and cysteine sulfinate (Ki = 13.3 +/- 2.3 microM). Addition of extrasynaptosomal cysteate, aspartate, or cysteine sulfinate to synaptosomes loaded with [35S]cysteate induced rapid efflux of the cysteate. This efflux occurred via stoichiometric exchange of amino acids with half-maximal rates at 5.0 +/- 1.1 microM aspartate or 8.0 +/- 1.3 microM cysteine sulfinate. Conversely, added extrasynaptosomal cysteate exchanged for endogenous aspartate and glutamate with half-maximal rates at 5.0 +/- 0.4 microM cysteate. In the steady state after maximal accumulation of cysteate, the intrasynaptosomal cysteate concentrations exceeded the extrasynaptosomal concentrations by up to 10,000-fold. The measured concentration ratios were the same, within experimental error, as those for aspartate and glutamate. Depolarization, with either high [K+] or veratridine, of the plasma membranes of synaptosomes loaded with cysteate caused parallel release of cysteate, aspartate, and glutamate. It is concluded that neurons transport cysteate, cysteine sulfinate, aspartate, and glutamate with the same transport system. This transport system catalyzes homoexchange and heteroexchange as well as net uptake and release of all these amino acids.     

Utilization of sulfonates as sole sulfur source by soil bacteria including Comamonas acidovorans

Bacteria able to use cysteate, taurine or isethionate as sole source of carbon and energy were isolated from the soil. Tests of sulfur assimilation showed that sulfonate sulfur and sulfate sulfur supported comparable cell yields. Methanesulfonate, 1-dodecanesulfonate and p-toluenesulfonate also served as sole source of sulfur for strain I91, identified as Comamonas (Pseudomonas) acidovorans. Competition studies with strain I91 showed that the presence of sulfate inhibits cysteate, isethionate or taurine incorporation. Pseudomonas aeruginosa PAO1, Comamonas acidovorans 14 and 105, and Acidovorax (Pseudomonas) facilis 332 used cysteate, isethionate, or taurine as sole source of sulfur while P. aeruginosa PAO716 and PAO718 used only taurine.     

Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Summary. Among the amino acids produced by plants cysteine plays a special role as a mediator between assimilatory sulfate reduction and provision of reduced sulfur for cell metabolism. Part of this characteristic feature is the presence of cysteine synthesis in plastids, mitochondria and cytosol. Plants are the major source of reduced sulfur for human and animal nutrition. Cysteine biosynthesis deserves special attention, since reduced sulfur is channelled from cysteine into many sulfur-containing compounds in food and feed. Recent investigations are reviewed that focus on structure and regulation of cysteine synthesis in the model plant Arabidopsis thaliana. These data indicate that cysteine synthesis is not just an intermediate reaction step but that it is part of a regulatory network that mediates between inorganic sulfur supply and the demand for reduced sulfur during plant growth and in response to environmental changes. Received December 3, 2001 Accepted December 21, 2001     

Biotin synthase is a pyridoxal phosphate-dependent cysteine desulfurase

Biotin synthase (BioB) is an iron-sulfur dimeric enzyme which catalyzes the last step in biotin synthesis. The reaction consists of the introduction of a sulfur atom into dethiobiotin. It is shown here that BioB displays a significant cysteine desulfurase activity, providing it with the ability to mobilize sulfur from free cysteine. This activity is dependent on pyridoxal 5'-phosphate (PLP) and dithiothreitol and proceeds through a protein-bound persulfide. Like other cysteine desulfurases, BioB binds 1 equiv of PLP. By site-directed mutagenesis, two conserved cysteines, Cys97 and Cys128, are shown to be critical for cysteine desulfuration and are good candidates as the site for a persulfide. Since biotin synthase activity is greatly increased by PLP and cysteine, even though it does not exceed 1 nmol of biotin/nmol of monomer, it is proposed that cysteine desulfuration is intimately linked to biotin synthesis. New scenarios for sulfur insertion into dethiobiotin, in which cysteine persulfides play a key role, are discussed.     

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine–aldimine complex to a substrate–aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.     

Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment.

The transsulfuration pathways allow the interconversion of homocysteine and cysteine with the intermediary formation of cystathionine. The various organisms studied up to now incorporate reduced sulfur into a three- or a four-carbon chain and use differently the transsulfuration pathways to synthesize sulfur amino acids. In enteric bacteria, the synthesis of cysteine is the first step of organic sulfur metabolism and homocysteine is derived from cysteine. Fungi are capable of incorporating reduced sulfur into a four-carbon chain, and they possess two operating transsulfuration pathways. By contrast, synthesis of cysteine from homocysteine is the only existing transsulfuration pathway in mammals. In Saccharomyces cerevisiae, genetic, phenotypic, and enzymatic study of mutants has allowed us to demonstrate that homocysteine is the first sulfur amino acid to be synthesized and cysteine is derived only from homocysteine (H. Cherest and Y. Surdin-Kerjan, Genetics 130:51-58, 1992). We report here the cloning of genes STR4 and STR1, encoding cystathionine beta-synthase and cystathionine gamma-lyase, respectively. The only phenotypic consequence of the inactivation of STR1 or STR4 is cysteine auxotrophy. The sequencing of gene STR4 has allowed us to compare all of the known sequences of transsulfuration enzymes and enzymes catalyzing the incorporation of reduced sulfur in carbon chains. These comparisons reveal a partition into two families based on sequence motifs. This partition mainly correlates with similarities in the catalytic mechanisms of these enzymes.     

Taurine serves as sole source of nitrogen for aerobic and anaerobic growth by <Emphasis Type="Italic">Klebsiella</Emphasis> sp.

The sulfur of taurine can be assimilated by Klebsiella sp. during aerobic growth, but not fermentative growth. However, taurine’s N can be utilized by this bacterium as sole nitrogen source for both aerobic and anaerobic growth. Two other amino-containing sulfonates (3-aminopropanesulfonate and cysteate) were also examined for their abilities to serve as nitrogen sources for Klebsiella sp. during the different growth conditions. The result shows that 3-aminopropanesulfonate only supports aerobic growth while cysteate does not under either condition.     

GLUTAMATE AND RELATED AMINO ACIDS IN CAT SPINAL ROOTS, DORSAL ROOT GANGLIA AND PERIPHERAL NERVES

Of the free amino acids found in extracts of cat spinal roots, dorsal root ganglia and peripheral nerves, only glutamate was present in disproportionately high concentrations in those parts of the dorsal roots between ganglia and spinal cord. This distribution suggests that the high dorsal root levels of glutamate may result from synthesis in dorsal root ganglia and subsequent transport towards the spinal cord. Four excitant amino acids were detected in the extracts: aspartate, cysteate, cysteine sulphinate and glutamate. The unique regional distribution of glutamate is consistent with the proposed role of this amino acid as an excitatory transmitter at the terminals of primary afferent fibres.     

Bacillus subtilis cysteine synthetase is a global regulator of the expression of genes involved in sulfur assimilation

The synthesis of L-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH, almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.     

Two transsulfurylation pathways in Klebsiella pneumoniae

In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-gamma-synthase and cystathionine-beta-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-beta-synthase and cystathionine-gamma-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.     

Stimulation of h(2)s emission from pumpkin leaves by inhibition of glutathione synthesis

The effect of inhibitors of glutathione (GSH) synthesis, namely gamma-methyl glutamic acid, d-glutamic acid, cystamine, methionine-S-sulfoximine (MSX), buthionine-S-sulfoximine, and GSH itself, on the emission of H(2)S was investigated. All these compounds stimulated H(2)S emission from pumpkin (Cucurbita pepo L. cv Small Sugar Pumpkin) leaf discs in response to sulfate. MSX and GSH were the most effective compounds, stimulating H(2)S emission from leaf discs of mature pumpkin leaves by about 80% in response to sulfate. Both inhibitors did not appreciably enhance H(2)S emission in response to l-cysteine and inhibited H(2)S emission in response to sulfite.Treatment with MSX or GSH enhanced the uptake of sulfate by pumpkin leaf discs, but did not affect the incorporation of sulfate into reduced sulfur compounds. Inhibition of GSH synthesis by MSX or GSH caused an increase in the pool size of cysteine, and, simultaneously, reduced the incorporation of labeled sulfate into cysteine. The incorporation of labeled sulfate into the sulfite and sulfide pools of the cells are stimulated under these conditions.These observations are consistent with the idea that inhibition of GSH synthesis leads to an elevated cysteine pool that inhibits further cysteine synthesis. The H(2)S emitted under these conditions appears to arise from diversion of a precursor of the sulfur moiety of l-cysteine. Therefore, stimulation of H(2)S emission in response to sulfate upon inhibition of GSH synthesis may reflect a role of H(2)S emission in keeping the cysteine concentration below a critical level.     

Role of the Liver in Regulation of Body Cysteine and Taurine Levels: A Brief Review

The first-pass metabolism of dietary sulfur amino acids by the liver and the robust upregulation of hepatic cysteine dioxygenase activity in response to an increase in dietary protein or sulfur amino acid level gives the liver a primary role in the removal of excess cysteine and in the synthesis of taurine. Hepatic taurine synthesis is largely restricted by the low availability of cysteinesulfinate as substrate for cysteinesulfinate decarboxylase, and taurine production is increased when cysteinesulfinate increases in response to an increase in the hepatic cysteine concentration and the associated increase in cysteine dioxygenase activity. The upregulation of cysteine dioxygenase in the presence of cysteine is a consequence of diminished ubiquitination of cysteine dioxygenase and a slower rate of degradation by the 26S proteasome.     

Dissimilation of C3-sulfonates

Cysteate and sulfolactate are widespread natural products in the environment, while propanesulfonate, 3-aminopropanesulfonate and propane-1,3-disulfonate are xenobiotics. While some understanding of the bacterial assimilation of cysteate sulfur has been achieved, details of the dissimilation of cysteate and sulfolactate by microbes together with information on the degradation of the xenobiotics have only recently become available. This minireview centres on bacterial catabolism of the carbon moiety in these C₃-sulfonates and on the fate of the sulfonate group. Three mechanisms of desulfonation have been established. Firstly, cysteate is converted via sulfopyruvate to sulfolactate, which is desulfonated to pyruvate and sulfite; the latter is oxidized to sulfate by a sulfite dehydrogenase and excreted as sulfate in Paracoccus pantotrophus NKNCYSA. Secondly, sulfolactate can be converted to cysteate, which is cleaved in a pyridoxal 5′-phosphate-coupled reaction to pyruvate, sulfite and ammonium ions; in Silicibacter pomeroyi DSS-3, the sulfite is excreted largely as sulfite. Both desulfonation reactions seem to be widespread. The third desulfonation mechanism is oxygenolysis of, e.g. propanesulfonate(s), about which less is known.     

Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady state conditions with limiting sulfate

The physiological roles of the transsulfuration and direct sulfhydration pathways in Chlorella sorokiniana growing under steady state photoautotrophic conditions with limiting sulfate were studied by following the patterns of assimilation of 35SO4(2-) into sulfur amino acids. The labeling patterns expected of each pathway were defined by means of models based on the rates of net synthesis of the terminal pools of GSH, protein cysteine, and protein methionine. The labeling patterns observed are entirely consistent with the transsulfuration pathway and inconsistent with the direct sulfhydration pathway. By analysis of the amounts of radioactivity present in key intermediates at labeling times as short as 1 s, it was demonstrated that direct sulfhydration makes no detectable contribution to homocysteine biosynthesis, and if operative contributes no more than approximately 3% of the total homocysteine biosynthesized. From the combined determinations of the initial rates of labeling and net rates of synthesis of the various sulfur amino acids, a tentative working model is presented that summarizes our best current estimates of the major fluxes of sulfur in the experimental system. The labeling data further showed that soluble cysteine consists of at least two pools. One pool, termed "rapidly turning over" cysteine comprises less than 1% of the total soluble cysteine, and is the precursor of GSH, protein cysteine, and, almost certainly, cystathionine. The other pool, "slowly turning over" cysteine, appears to be in equilibrium with "rapidly turning over" cysteine, but not to be further metabolized.     

Synthesis of the sulfur amino acids: cysteine and methionine

This review will assess new features reported for the molecular and biochemical aspects of cysteine and methionine biosynthesis in Arabidopsis thaliana with regards to early published data from other taxa including crop plants and bacteria (Escherichia coli as a model). By contrast to bacteria and fungi, plant cells present a complex organization, in which the sulfur network takes place in multiple sites. Particularly, the impact of sulfur amino-acid biosynthesis compartmentalization will be addressed in respect to localization of sulfur reduction. To this end, the review will focus on regulation of sulfate reduction by synthesis of cysteine through the cysteine synthase complex and the synthesis of methionine and its derivatives. Finally, regulatory aspects of sulfur amino-acid biosynthesis will be explored with regards to interlacing processes such as photosynthesis, carbon and nitrogen assimilation.     

Genetic Analysis of a New Mutation Conferring Cysteine Auxotrophy in Saccharomyces Cerevisiae: Updating of the Sulfur Metabolism Pathway

We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine. The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity. This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine. The phenotype induced by this mutation implies that, in S. cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine. This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S. cerevisiae. The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1-1 mutation undergoes a particularly high frequency of meiotic gene conversion.