Glutamine metabolism in isolated perfused rat liver. The transamination pathway

In isolated perfused rat liver, added 4-methyl-thio-2-oxobutyrate and phenylpyruvate are rapidly transaminated to the corresponding amino acids with glutamine, the latter being supplied via the portal vein or by endogenous synthesis. With portal glutamine concentrations below 5mM and in the presence of a oxo-acid acceptor, the flux through glutamine transaminases exceeded the ammonium ion-stimulated glutaminase flux. 4-Methylthio-2-oxobutyrate-induced extra glutamine uptake was not dependent on the perfusate pH in the range of pH 7 to 8. During glutamine/4-methylthio-2-oxobutyrate transamination, the amide nitrogen of glutamine is fully recovered as glutamate, ammonia, urea and alanine. Oxoglutarate formed by omega-amidase activity is released as glutamate or oxidized by oxoglutarate dehydrogenase. alpha-Cyanocinnamate, the inhibitor of the monocarboxylate translocator in the mitochondrial membrane inhibited 4-methylthio-2-oxobutyrate-induced glutamine uptake and methionine release by about 30%. This might indicate that about 2/3 of glutamine transaminase flux is cytosolic. alpha-Cyanocinnamate inhibited 4-methylthio-2-oxobutyrate-induced glutamate efflux by about 90%. Stimulation of flux through glutamine transaminases is accompanied by a 70-80% inhibition of glutaminase flux. This is not explained by a direct inhibition of glutaminase by 4-methylthio-2-oxobutyrate but by a substrate competition between glutaminase and glutamine transaminases. 4-Methylthio-2-oxobutyrate decreases glutamine release by the liver due to withdrawal by transamination. The oxo acid itself is without effect on glutamine synthetase flux. With respect to hepatocyte heterogeneity there is no evidence for a zonal distribution of glutamine transaminase activities, as it has been shown for glutamine synthetase and glutaminase activities.     

Functional Identification of APIP as Human mtnB,a Key Enzyme in the Methionine Salvage Pathway

The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Crystal structures of aminotransferases Aro8 and Aro9 from Candida albicans and structural insights into their properties

Aminotransferases catalyze reversibly the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5′-phosphate (PLP) as a cofactor. Various aminotransferases acting on a range of substrates have been reported. Aromatic transaminases are able to catalyze the transamination reaction with both aromatic and acidic substrates. Two aminotransferases from C. albicans, Aro8p and Aro9p, have been identified recently, exhibiting different catalytic properties. To elucidate the multiple substrate recognition of the two enzymes we determined the crystal structures of an unliganded CaAro8p, a complex of CaAro8p with the PLP cofactor bound to a substrate, forming an external aldimine, CaAro9p with PLP in the form of internal aldimine, and CaAro9p with a mixture of ligands that have been interpreted as results of the enzymatic reaction. The crystal structures of both enzymes contains in the asymmetric unit a biologically relevant dimer of 55?kDa for CaAro8 and 59?kDa for CaAro9p protein subunits. The ability of the enzymes to process multiple substrates could be related to a feature of their architecture in which the active site resides on one subunit while the substrate-binding site is formed by a long loop extending from the other subunit of the dimeric molecule. The separation of the two functions to different chemical entities could facilitate the evolution of the substrate-binding part and allow it to be flexible without destabilizing the conservative catalytic mechanism.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

家蚕体内因缺乏维生素B6而引起的若干代谢变动

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori 5龄幼虫,探讨了缺乏维生素B₆（VB₆）对蚕体氨基酸代谢、脂肪酸代谢以及转氨酶活力的影响。缺乏VB₆引起支链氨基酸分解代谢受阻,幼虫体液中大量积累亮氨酸、缬氨酸和异亮氨酸。同时因绢丝腺发育停滞,丝氨酸也在体液中积累。另一方面,缺乏VB₆幼虫体液中赖氨酸、脯氨酸、精氨酸、甲硫氨酸和谷氨酸含量减少,其中赖氨酸尤为突出。推测缺乏VB₆引起赖氨酸分解代谢亢进。结果还表明,缺乏VB₆幼虫体内脂肪酸代谢异常,谷丙转氨酶活力显著低下。     

The role of free amino acids in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio

The present study investigated (1) the free amino acid (FAA) composition in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio, (2) enzyme systems involved in amino acid metabolism and (3) the effect of amino acids on sperm viability under in vitro storage conditions. In the seminal plasma of O. mykiss, the main FAAs were arginine, glutamic acid, isoleucine, leucine, methionine and proline, in spermatozoa cysteine, arginine and methionine. In the seminal plasma of C. carpio, the main FAAs were alanine, arginine, cysteine, glutamic acid, histidine, leucine, lysine, methionine and proline, in spermatozoa arginine, glutamic acid, histidine, leucine and lysine. When spermatozoa were incubated for 48 h together with the seminal plasma, the quantitative amino acid pattern changed in both species indicating their metabolism. In spermatozoa and seminal plasma of O. mykiss and C. carpio, the following enzymes were found to be related to amino acid metabolism: transaminases (specific for alanine, aspartate, isoleucine and leucine), decarboxylases (specific for valine and lysine), glutamate dehydrogenase and α‐keto acid dehydrogenases (substrates: 3‐methyl‐2‐oxovaleric acid and 4‐methyl‐2‐oxovalerate). These data demonstrate that amino acid catabolism by transamination, decarboxylation and oxidative deamination can occur in semen of the two species. Also activity of methionine sulphoxide reductase was detected, an enzyme which reduces methionine sulphoxide to methionine. This reaction plays an important role in antioxidant defence. To determine the effect of FAAs on the sperm viability, C. carpio and O. mykiss spermatozoa were incubated in sperm motility inhibiting saline solution containing different amino acids. Methionine had a positive effect on the sperm viability in both species. Taken together this result with the in vivo occurrence of methionine and of methionine reductase in semen, it can be assumed that this amino acid plays an important role in antioxidant defence. Also isoleucine in O. mykiss and leucine in C. carpio had a positive effect on sperm viability. As seminal plasma and spermatozoa of the two species exhibit enzyme activities to catabolize leucine and isoleucine, they might serve as additional energy resources especially during prolonged incubation and storage periods.     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

Effect of 24-hour starvation on amino acid pool composition and enzyme activities of rat brown adipose tissue

The effect of 24-hr starvation on the amino acid pool composition and its concentration ratios with respect to blood and plasma as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase have been studied in rat brown adipose tissue. Starvation induced a considerable decrease of pool amino acid concentration. Alanine and taurine were the amino acids in which the decrease was more marked. Small changes were observed in the activities of the enzymes studied, with decreases only in glutamate dehydrogenase and adenylate deaminase. These changes agree with a decrease in amino acid utilization in this tissue induced by starvation.     

Methionine metabolism by rat muscle and other tissues. Occurrence of a new carnitine intermediate.

Perfused rat hindquarter preparations were shown to incorporate radioactivity from [U-14C]methionine into citrate-cycle intermediates, lactate, alanine, glutamate, glutamine and CO2. During perfusion, large amounts of methionine were also oxidized to methionine sulphoxide. The capacity for transamination of methionine or its oxo analogue, 4-methylthio-2-oxobutyrate, by muscle extracts was demonstrated. Rat skeletal muscle, heart, liver and kidney mitochondria, when incubated with the latter plus radiolabelled carnitine, formed a newly identified carnitine derivative, 3-methylthiopropionylcarnitine. It is concluded that the capacity for oxidation of methionine by a trans-sulphuration-independent pathway occurs in several mammalian tissues. The extent of inter-organ handling of intermediates in this pathway(s) is discussed.     

S-Methylmethionine Conversion to Dimethylsulfoniopropionate: Evidence for an Unusual Transamination Reaction

Leaves of Wollastonia biflora (L.) DC. synthesize the osmoprotectant 3-dimethylsulfoniopropionate (DMSP) from methionine via S-methylmethionine (SMM) and 3-dimethylsulfoniopropionaldehyde (DMSP-ald); no other intermediates have been detected. To test whether the amino group of SMM is lost by transamination or deamination, [methyl-2H3,15N]SMM was supplied to leaf discs, and 15N-labeling of amino acids was monitored, along with synthesis of [2H3]DMSP. After short incubations more 15N was incorporated into glutamate than into other amino acids, and the 15N abundance in glutamate exceeded that in the amide group of glutamine (Gln). This is more consistent with transamination than deamination, because deamination would be predicted to give greater labeling of Gln amide N due to reassimilation, via Gln synthetase, of the 15NH4+ released. This prediction was borne out by control experiments with 15NH4Cl. The transamination product of SMM, 4-dimethylsulfonio-2-oxobutyrate (DMSOB), is expected to be extremely unstable. This was confirmed by attempting to synthesize it enzymatically from SMM using L-amino acid oxidase or Gln transaminase K and from 4-methylthio-2-oxobutyrate using methionine S-methyltransferase. In each case, the reaction product decomposed rapidly, releasing dimethylsulfide. The conversion of SMM to DMSP-ald is therefore unlikely to involve a simple transamination that generates free DMSOB. Plausible alternatives are that DMSOB is channeled within a specialized transaminase-decarboxylase complex or that it exists only as the bound intermediate of a single enzyme catalyzing an unusual transamination-decarboxylation reaction.     

Metabolism of amino acids in Ascaridia galli: transamination

Ascaridia galli, using 2-oxoglutarate as an acceptor, transaminates alanine and aspartate at significantly high rates. Among other amino acids valine, phenylalanine, leucine, isoleucine, arginine, tyrosine and methionine are metabolised at moderate rates while lysine, serine, threonine, cysteine, glycine, histidine, tryptophan, DOPA and GABA appear to be inert in this respect. Body parts mimic the whole worm in the pattern of transamination of various amino acids with the exception of methionine. Alanine, aspartate and glutamate may transfer their amino group also to pyruvate and oxaloacetate. Alanine and aspartate: 2-oxoglutarate transaminases are located mainly in the cytosol and mitochondrial fractions.     

Characterization of Glycogen and Amino Acid Pool of Entamoeba histolytica by 13C-NMR Spectroscopy

ABSTRACT. Trophozoite extracts of axenic Entamoeba histolytica were investigated by natural-abundance ¹³C-NMR spectroscopy. The extracts were found to contain a high level of glycogen (30 mM glucose equivalents), which had a compact structure as suggested by α (1 → 6) branch points every 5–6 glucose residues. As other major metabolites, we identified putrescine (9.5 mM) and the following free amino acids: tyrosine and phenylalanine (1 mM), glycine, lysine and methionine (2 mM), isoleucine (5 mM), proline and valine (6–7 mM), leucine (11 mM) and glutamate (22 mM). Glutamate and proline may serve, together with putrescine, as intracellular osmolytes.     

Effect of cold-temperature exposure and acclimation on amino acid pool changes and enzyme activities of rat brown adipose tissue

The amino acid pool composition and its concentration ratios with respect to blood and plasma, as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamine synthetase, adenylate deaminase and glutamate dehydrogenase have been studied in the interscapular brown adipose tissue of control, 12-h cold-exposed and 15-day cold-acclimated rats. Cold temperature affected the amino acid metabolism and pool composition more intensely after 15 days than after 12-h cold-exposure, even though the patterns of change were very similar in both groups. Cold temperatures induced a decrease in glutamine and an increase in glutamate concentration in the tissue. This probably increased the metabolism of branched chain amino acids and caused a decrease in adenylate deaminase activity. It also seemed to increase alanine utilization. We concluded that amino acid metabolism in brown adipose tissue is enhanced by cold temperature acclimation.     

Growth and spore germination factors of various strains of Bacillus sphaericus

The work was aimed at studying the requirements of sixteen Bacillus sphaericus strains with a different larvicidal activity in amino acids and some other compounds necessary for their growth and spore germination. Most of the strains were found to require arginine, glutamate, methionine, threonine, serine, glycine, alanine and lysine, but they did not assimilate phenylalanine and proline. Arginine, methionine and glutamate were shown to be the most effective inductors of spore germination. Specific differences were detected in the requirements of virulent and avirulent strains. Glucose repressed both spore germination and spore formation.     

MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila

To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.     

Mechanistic diversity in the RuBisCO superfamily: the "enolase" in the methionine salvage pathway in Geobacillus kaustophilus

D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the d-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-d-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this "enolase" is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO- and "enolase"-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the "enolase" reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the "enolases" from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from d-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the "enolase"-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated "enolase" from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal alpha+beta domain instead of the C-terminal (beta/alpha)8-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all "enolases", is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus "enolase" is unable to catalyze the "enolase" reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.     

RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme in the fixation of CO(2) in the Calvin cycle of plants. Many genome projects have revealed that bacteria, including Bacillus subtilis, possess genes for proteins that are similar to the large subunit of RuBisCO. These RuBisCO homologues are called RuBisCO-like proteins (RLPs) because they are not able to catalyse the carboxylase or the oxygenase reactions that are catalysed by photosynthetic RuBisCO. It has been demonstrated that B. subtilis RLP catalyses the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. The structure of DK-MTP-1-P is very similar to that of ribulose-1,5-bisphosphate (RuBP) and the enolase reaction is a part of the reaction catalysed by photosynthetic RuBisCO. In this review, functional and evolutionary relationships between B. subtilis RLP of the methionine salvage pathway, other RLPs, and photosynthetic RuBisCO are discussed. In addition, the fundamental question, 'How has RuBisCO evolved?' is also considered, and evidence is presented that RuBisCOs evolved from RLPs.     

Mechanistic diversity in the RuBisCO superfamily: a novel isomerization reaction catalyzed by the RuBisCO-like protein from Rhodospirillum rubrum

Some homologues of D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) do not catalyze carboxylation and are designated RuBisCO-like proteins (RLPs). The RLP from Rhodospirillum rubrum (gi:83593333) catalyzes a novel isomerization reaction (overall 1,3-proton transfer reaction; likely, two 1,2-proton transfer reactions) that converts 5-methylthio-D-ribulose 1-phosphate to a 3:1 mixture of 1-methylthioxylulose 5-phosphate and 1-methylthioribulose 5-phosphate. Disruption of the gene encoding the RLP abolishes the ability of R. rubrum to utilize 5'-methylthioadenosine as a sole sulfur source, implicating a new, as-yet-uncharacterized, pathway for sulfur salvage.