Dissimilation of methionine by fungi

Soil fungi that attacked methionine required a utilizable source of energy such as glucose for growth. This is an example of co-dissimilation. Experiments with one of the fungi, representative of the group, are reported. In the absence of glucose, pregrown mycelium, even when depleted of energy reserves, oxidatively deaminated methionine with accumulation of α-keto-γ-methyl mercapto butyric acid and α-hydroxy-γ-methyl mercapto butyric acid. When glucose was provided, all of the sulfur of methionine was released as methanethiol, part of which was oxidized to dimethyl disulfide. No sulfate, sulfide, or hydrosulfide products were detected. Evidence was obtained that deaminase and demethiolase were constitutive. Deamination preceded demethiolation and α-keto butyric acid accumulated as a product of the two reactions. Other carbon residues were α-hydroxy butyric acid and α-amino butyric acid. Inability of the fungus to metabolize α-keto butyrate was responsible for its inability to utilize methionine as a source of carbon and energy. Several other fungi isolated from soil grew on α-amino butyrate but could not grow on methionine owing to inability to demethiolate it.     

In Vitro and In Vivo Oxidation of Methionine Residues in Small, Acid-Soluble Spore Proteins from Bacillus Species

Methionine residues in α/β-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H₂O₂). These oxidized α/β-type SASP no longer bound to DNA effectively, but DNA binding protected α/β-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized α/β-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the α/β-type SASP’s ability to bind to DNA. Both tBHP and H₂O₂ caused some oxidation of the two methionine residues of an α/β-type SASP (SspC) in spores of Bacillus subtilis, although one methionine which is highly conserved in α/β-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H₂O₂ than were wild-type spores. The major mechanism operating for dealing with oxidative damage to α/β-type SASP in spores is DNA binding, which protects the protein’s methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since α/β-type SASP containing oxidized methionine residues no longer bind DNA well and α/β-type SASP-DNA binding is essential for long-term spore survival.     

Metabolism of propionate by sheep liver. Oxidation of propionate by homogenates

1. The rate and stability to aging of the metabolism of propionate by sheep-liver slices and sucrose homogenates were examined. Aging for up to 20min. at 37° in the absence of added substrate had little effect with slices, whole homogenates or homogenates without the nuclear fraction. 2. Metabolism of propionate by sucrose homogenates was confined to the mitochondrial fraction, but the mitochondrial supernatant (microsomes plus cell sap) stimulated propionate removal. 3. The rate of propionate metabolism by liver slices was higher in a high potassium phosphate–bicarbonate medium [0·88(±s.e.m. 0·16)μmole/mg. of N/hr.] than in Krebs–Ringer bicarbonate medium [0·44(±s.e.m. 0·13)μmole/mg. of N/hr.]. 4. Metabolism of propionate by sucrose homogenates freed from nuclei was dependent on the presence of oxygen, carbon dioxide and ATP. Propionate removal was stimulated 250% by Mg²⁺ ions and 670% by cytochrome c. 5. In the complete medium 2·39(±s.e.m. 0·15)μmoles of propionate were consumed/mg. of N/hr. 6. The ratio of oxygen consumption to propionate utilization was sufficient to account for the complete oxidation of half the propionate consumed. 7. The only products detected under these conditions were succinate, fumarate and malate. Propionate had no effect on the production of lactate from endogenous sources and did not itself give rise to lactate. 8. Methylmalonate did not accumulate when propionate was metabolized and was not oxidized. It was detected as an intermediate in the conversion of propionyl-CoA into succinate. The rate of this reaction sequence was adequate to account for the rate of propionate metabolism by sucrose homogenates or slices, provided that the rate of formation of propionyl-CoA was not limiting. 9. The methylmalonate pathway was predominantly a mitochondrial function. 10. The metabolism of propionate appeared to be dependent on active oxidative phosphorylation.     

Sulfur-Containing Amino Acids as Precursors of Thiols in Anoxic Coastal Sediments

Sulfur-containing amino acids were examined as precursors for thiols in anoxic coastal sediments. Substrates (10 to 100 μM) were anaerobically incubated with sediment slurries; thiols were assayed as isoindole derivatives by high-performance liquid chromatography; and microbial transformations of thiols, in contrast to their chemical binding by sediment particles, were identified by inhibition with a mixture of chloramphenicol and tetracycline. Methionine and homocysteine were transformed to methanethiol and 3-mercaptopropionate (3-MPA); methionine stimulated mainly methanethiol production, whereas homocysteine generated more 3-MPA than methanethiol. 2-Keto-4-methiolbutyrate yielded results similar to those with methionine, indicating that demethiolation yields methanethiol at the keto-acid level. Glutathione gave rise to cysteine, which was further transformed to 3-mercaptopyruvate and thence to mercaptoacetate and mercaptoethanol. Mercaptoethanol was oxidized to mercaptoacetate, which was biologically consumed. In conclusion, sulfurcontaining amino acids contribute to the range of thiols that occur in anoxic coastal sediments. New metabolic and environmental transformations were identified: the production of 3-MPA as a metabolite of methionine and the transformation of mercaptopyruvate to mercaptoethanol and mercaptoacetate.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Inhibition of Sterol Biosynthesis in Chlorella sorokiniana by Triparanol

When Chlorella sorokiniana was cultured in the presence of 1 mg/1 triparanol succinate, there was a 42% reduction in total sterol concentration. Algal biomass was reduced by approximately the same amount. In addition to the cycloartenol, cyclolaudenol, 24-methyl-pollinastanol, ergosta-5, 7-dien-3β-ol, and ergosterol that occur in control culture, pollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergosta-8, 14, 22-trien-3β-ol, 5α-ergosta-8(14), 22-dien-3β-ol, 5α-ergosta-8(9), 22-dien-3β-ol, 5α-ergosta-8, 14-dien-3β-ol, 5α-ergost-8(9)-3n-3β-ol, 5α-ergost-8(14)-en-3β-ol, 5α-ergosta-7, 22-dien-3β-ol, and 5α-ergost-7-en-3β-ol were isolated and identified from triparanol succinate-treated cells. A biosynthetic pathway for sterol biosynthesis in this organism is postulated based on all the sterols that were isolated and identified in triparanol-treated cultures of C. sorokiniana. Cyclolaudenol appears to be the product of the first alkylation at C-24 in this organism rather than the more common 24-methylene cycloartanol. Since 24-methylene sterols are needed for the second alkylation reaction, this would explain the absence of C-29 sterols in C. sorokiniana. Four of the sterols identified in C. sorokiniana are reported for the first time in a living organism. They are: 24-methyl pollinastanol, 5α-ergosta-8, 14, 22-trien-3β-ol, 5α-ergosta-8(14), 22-dien-3β-ol and 5α-ergost-8(14)-en-3β-ol.     

Studies on the biosynthesis of phenols in fungi. Production of 4-methoxytoluquinol, epoxysuccinic acid and a diacetylenic alcohol by surface cultures of Lentinus degener I.M.I. 110525

1. 4-Methoxytoluquinol was secreted into the medium by surface cultures of the basidiomycete Lentinus degener Kalchbr. (approx. 100mg./l. of medium). In addition, epoxysuccinic acid (150–200mg.) and a long-chain diacetylenic alcohol (3mg.) were also secreted. Epoxysuccinic acid has previously been found in the culture medium of some Fungi Imperfecti. These metabolites were all synthesized during the early phase of growth but maximum production occurred some time later. 2. Supplementation of the medium with cycloheximide or 8-azaguanine inhibited the production of epoxysuccinic acid. 3. Sodium [1-¹⁴C]acetate and 6-methyl[¹⁴C]salicylic acid were not incorporated into 4-methoxytoluquinol, but [U-¹⁴C]tyrosine and [Me-¹⁴C]methionine were incorporated to the extent of 0·55 and 4·75% respectively (minimum values). Degradation studies established that the aromatic ring and C-methyl group were derived from the ring and β-carbon atom of tyrosine; the O-methyl group alone was formed from methionine.     

Metabolic Basis for the Isoleucine, Pantothenate or Methionine Requirement of ilvG Strains of Salmonella typhimurium

Salmonella typhimurium strain DU501, which was found to be deficient in acetohydroxy acid synthase II (AHAS II) and to possess elevated levels of transaminase B and biosynthetic threonine deaminase, required isoleucine, methionine, or pantothenate for growth. This strain accumulated α-ketobutyrate and, to a lesser extent, α-aminobutyrate. We found that α-ketobutyrate was a competitive substrate for ketopantoate hydroxymethyltransferase, the first enzyme in pantothenate biosynthesis. This competition with the normal substrate, α-ketoisovalerate, limited the supply of pantothenate, which resulted in a requirement for methionine. Evidence is presented to support the conclusion that the ambivalent requirement for either pantothenate or methionine is related to a decrease in succinyl coenzyme A, which is produced from pantothenate and which is an obligatory precursor of methionine biosynthesis. The autointoxification by endogenously produced α-ketobutyrate could be mimicked in wild-type S. typhimurium by exogenously supplied α-ketobutyrate or salicylate, a known inhibitor of pantothenate biosynthesis. The accumulation of α-ketobutyrate was initiated by the inability of the residual AHAS activity provided by AHAS I to efficiently remove the α-ketobutyrate produced by biosynthetic threonine deaminase. The accumulation of α-ketobutyrate was amplified by the action of transaminase B, which decreased the isoleucine pool by catalyzing the formation of α-keto-β-methylvalerate and aminobutyrate from isoleucine and α-ketobutyrate; this resulted in release of threonine deaminase from end product inhibition and unbridled production of α-ketobutyrate. Isoleucine satisfied the auxotrophic requirement of the AHAS II-deficient strain by curtailing the activity of threonine deaminase. Additional lines of evidence based on genetic and physiological experiments are presented to support the basis for the autointoxification of strain DU501 as well as other nonpolarigenic ilvG mutant strains.     

Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis

Euglena gracilis cells synthesize the key tetrapyrrole precursor, δ-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain α-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, α-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and α-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP⁺, and was inhibited by NAD⁺. Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of α-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.     

The periodate oxidation of amino acids with reference to studies on glycoproteins

1. All α-amino acids are oxidized by periodate, but at different rates. 2. The rates of oxidation of individual α-amino acids vary with pH. In general, oxidation proceeds more rapidly at alkaline pH. 3. Serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine and histidine are rapidly and extensively oxidized by periodate. 4. Cysteine, cystine, methionine, tryptophan, tyrosine and histidine are oxidized by periodate when they are substituted in the carboxyl and amino groups, as in a polypeptide chain.     

Fibrin Clot Structure and Mechanics Associated with Specific Oxidation of?Methionine Residues in Fibrinogen

Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels.     

Microbial Reduction of 1,3-Dioxo-2-Methyl-2-(3′-Oxo-6′-Carbomethoxyhexyl)-Cyclopentane to Form 1β-Hydroxy-3-Oxo-2β-Methyl-2α-(3′-Oxo-6′-Carbomethoxy-hexyl)-Cyclopentane, an Intermediate for Steroid Total Synthesis

The rate and extent of stereoselective reduction of 1,3-dioxo-2-methyl-2-(3′-oxo-6′-carbomethoxyhexyl)-cyclopentane to form the 1β-hydroxy-2β-methyl isomer by cultures of Schizosaccharomyces pombe ATCC 2476 was dramatically increased by addition to the fermentation of certain α,β-unsaturated ketones and allyl alcohol.     

Use of 13C Nuclear Magnetic Resonance and Gas Chromatography To Examine Methionine Catabolism by Lactococci

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, ¹³C nuclear magnetic resonance (NMR) performed with uniformly enriched [¹³C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the ¹³C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.     

In Vivo and In Vitro Studies on gamma-Aminobutyric Acid Metabolism with the Radish Plant (Raphanus sativus, L.)

Labeled glutamate was rapidly converted to γ-aminobutyrate in intact, excised radish (Raphanus sativus L., var. Champion) leaves. Labeled γ-aminobutyrate was metabolized via succinate and the Krebs cycle and was not carboxylated to form glutamate. Administration of carbon-14 and tritium-labeled succinate indicated that less than 10% of the γ-aminobutyrate formation occurs by amination of succinic semialdehyde. Therefore, most γ-aminobutyrate formation must be via glutamate decarboxylation.     

Conversion of Methionine to Thiols by Lactococci,Lactobacilli, and Brevibacteria

Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and α-ketoglutarate was used as the amino group acceptor. In Lactococcus lactis subsp. cremoris S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of l-methionine γ-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix.The primary classes of compounds that contribute to cheese flavor include amino acids and their degradation products, peptides, carbonyl compounds, and fatty acids. These partition primarily into the aqueous fraction of cheese (3). The volatile fraction of cheese has sulfur-containing compounds such as methanethiol, methional, dimethyl sulfide, dimethyl tetrasulfide, carbonyl sulfide, and hydrogen sulfide (28), and they contribute to the aroma of cheese (7). Methanethiol has been associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2), and it is also implicated as an influential aroma and flavor compound in many foods, including surface-ripened cheeses that use brevibacteria (17). However, methanethiol, when present alone, does not contribute to typical Cheddar-like flavor notes in cheese (17).Production of methanethiol is important in cheese, but the Met biosynthetic and catabolic pathways vary among bacteria (23). The mechanisms involved and amounts of methanethiol produced during cheese ripening also vary. In an effort to increase and accelerate the development of typical Cheddar cheese flavor, adjunct bacteria have been used during the manufacture of low- and full-fat cheese. Initial selection of flavor adjunct cultures focused on those bacteria used to accelerate flavor development in full-fat cheese, which are typically lactobacilli because they dominate (10⁷ to 10⁹ CFU/g of cheese during storage at 8°C) the microflora during aging (15). The Lactobacillus genus is considered to be a member of the nonstarter lactic acid bacteria subgroup because it is not added with the starter culture for Cheddar cheese. In addition to lactobacilli, micrococci and pediococci have been used as adjunct bacteria to aid in flavor development (20). Brevibacteria, which are normally found on the surfaces of Limburger and other Trappist-type cheeses, are not traditionally used as flavor adjuncts in Cheddar cheese. One advantage these organisms have over other adjuncts is their profuse production of methanethiol (8). Weimer et al. (30) successfully used Brevibacterium linens as an adjunct to improve the flavor of low-fat Cheddar cheese.The mechanism for the production of methanethiol in cheese by bacteria can be a result of the direct catabolism of Met or it can arise from inadvertent catalysis by other enzymes (1, 6, 17). The most direct route to methanethiol is the conversion of Met to methanethiol, ammonia, and α-ketobutyrate (Fig. ​(Fig.1).1). This transformation is catalyzed by inducible Met γ-lyase, a pyridoxal phosphate (PLP)-dependent enzyme (24) which has been purified to homogeneity from Pseudomonas putida (14, 26), Aeromonas spp. (27), and Clostridium sporogenes (16) and partially purified from B. linens (6). Open in a separate windowFIG. 1Metabolic pathways for Met interconversion. The primary intermediates and enzymes are listed. Enzyme 1 is cystathionine γ-lyase, enzyme 2 is cystathionine β-lyase, enzyme 3 is cystathionine β-synthase, enzyme 4 is homocysteine methyltransferase, enzyme 5 is aromatic aminotransferase (tyrB) or transaminase B (ilvE), enzyme 6 is amino acid oxidase, enzyme 7 is Met adenosyltransferase, and enzyme 8 is Met γ-lyase (adapted from reference 18 and 23).Pathways leading away from Met are important to consider because this amino acid is central to many other critical metabolic functions (Fig. ​(Fig.1).1). Utilization of Met for other metabolic functions would lower the pool of Met available for conversion to methanethiol. Methionine adenosyltransferase (S-adenosylmethionine [SAM] synthetase) converts Met into SAM at the expense of one ATP. SAM, one of the major methylating agents in a cell, is also important in the regulation of several of the Met-biosynthetic enzymes (22). Reduced SAM synthetase activity leads to low intracellular levels of SAM, resulting in the induction of the Met-biosynthetic pathway (32).Another mechanism that directs Met away from methanethiol is the deamination reaction to form α-keto γ-methyl thiobutyrate (KMTB). This conversion can be catalyzed by various aminotransferases (33) or amino acid oxidases (21). These enzymes are common in bacteria and are usually the last step in amino acid synthetic pathways (13). Amino acid oxidase activity is a possible route for KMTB production, and it is a possible route for subsequent methanethiol production in cheese, but this is unlikely because cheese tends to be anaerobic. Evidence for the conversion of KMTB to methanethiol is lacking for bacteria; however, this reaction has been shown to take place enzymatically in fungal species (23).When the catabolic pathways for Met are considered, the enzymes involved in the biosynthesis of Met must also be included. Although the principal reactions that these enzymes catalyze are involved in the synthesis of Met, they also coincidentally catalyze catabolic reactions that lead to the production of methanethiol and possibly other cheese flavor compounds. For example, cystathionine β-lyase, which primarily catalyzes the conversion of cystathionine to homocysteine, a reaction involved in the synthesis of Met (29), also catalyzes the conversion of Met to methanethiol, ammonia, and α-ketobutyrate but with 100 times less efficiency than that of its conversion to homocysteine in Lactococcus lactis subsp. cremoris S2 (1). This enzyme was purified from lactococci and has been implicated in the generation of methanethiol in Cheddar cheese (1). Cystathionine γ-lyase catalyzes the α,γ elimination of cystathionine to produce cysteine (Cys), α-ketobutyrate, and ammonia (19). A cystathionine γ-lyase purified from L. lactis subsp. cremoris is capable of catalyzing the α,γ elimination of Met to produce methanethiol at an efficiency much lower than that of the primary reaction it catalyzes (4). These enzymes may be present in the cells and liberated when the cells die and lyse during cheese storage, as occurs in Cheddar cheese ripening (10). With these observations in mind, the objective of this study was to determine the conversion pathways of Met to free thiols under laboratory and cheese-like conditions in bacteria used as starter cultures and flavor adjuncts in Cheddar cheese.     

Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2

l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its K_m for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min⁻¹ mg⁻¹. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Methanethiol is associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2, 27). The mechanism for the production of methanethiol in cheese is unknown, but it is linked to the catabolism of methionine (1, 15). l-Methionine γ-lyase (EC 4.4.1.11; MGL), also known as methionase, l-methionine γ-demethiolase, and l-methionine methanethiollyase (deaminating), is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of l-methionine to α-ketobutyrate, methanethiol, and ammonia by an α,γ-elimination reaction (26). It does not catalyze the conversion of d enantiomers (24–26). MGL in Pseudomonas putida is a multifunctional enzyme system since it catalyzes the α,γ- and α,β-elimination reactions of methionine and its derivatives (24). In addition, the enzyme also catalyzes the β-replacement reactions of sulfur amino acids (24). Since its discovery in Escherichia coli and Proteus vulgaris by Onitake (19), this enzyme has been found in various bacteria and is regarded as a key enzyme in the bacterial metabolism of methionine. However, this enzyme has not been purified to homogeneity from any food-grade microorganisms.MGL is widely distributed in bacteria, especially in pseudomonads, and is induced by the addition of l-methionine to the culture medium (9, 28). The enzyme has been purified from Pseudomonas putida (25), Aeromonas sp. (26), Clostridium sporogenes (11), and Trichomonas vaginalis (16) and partially purified from and characterized for Brevibacterium linens NCDO 739 (4).B. linens is a nonmotile, non-spore-forming, non-acid-fast, gram-positive coryneform bacterium normally found on the surfaces of Limburger and other Trappist-type cheeses. This organism tolerates salt concentrations ranging between 8 and 20% and is capable of growing in a broad pH range from 5.5 to 9.5, with an optimum pH of 7.0 (20). In Trappist-type cheeses, brevibacteria depend on Saccharomyces cerevisiae to metabolize lactate, which increases the pH of the curd, as well as to produce growth factors that are important for their growth (20). Interest in B. linens has focused around its ability to produce an extracellular protease, which has recently been isolated (21), and its ability to produce high levels of methanethiol (3, 9, 10, 22).B. linens produces various sulfur compounds, including methanethiol, that are thought to be important in Cheddar-like flavor and aroma (3, 9, 10, 22). Ferchichi et al. (9) suggested that MGL is responsible for the methanethiol-producing capability of B. linens but did not provide definitive evidence. Weimer et al. (28) proposed that B. linens BL2 is responsible for Cheddar-type flavor development in low-fat cheese, but again conclusive evidence was lacking. In this study, MGL was purified to homogeneity from B. linens BL2 and its physical and chemical properties were examined.     

The chemical synthesis and biological oxidation of 7α-hydroxy[26-14C]cholesterol, 7-dehydro[26-14C]cholesterol and 26-hydroxy[26-14C]cholesterol

1. 26-Hydroxycholesterol was obtained by reducing the methyl ester of (±)-3β-hydroxycholest-5-en-26-oic acid, which was synthesized from 25-oxonorcholesterol. 2. Methods for preparing 7α-hydroxycholesterol and 7-dehydrocholesterol were modified to allow the micro-scale preparation of these [¹⁴C]sterols from [26-¹⁴C]-cholesterol. 3. 26-Hydroxycholesterol was oxidized more readily than 7α-hydroxycholesterol, 7-dehydrocholesterol or cholesterol by mitochondrial preparations from livers of mice, rats, guinea pigs, common toads (Bufo vulgaris) and Caiman crocodylus. 4. (±)-3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was oxidized very rapidly to ¹⁴CO₂ by mouse and guinea-pig mitochondria without evident discrimination between the two optical isomers. 5. An enzyme system that oxidizes 26-hydroxycholesterol to 3β-hydroxycholest-5-en-26-oic acid was identified in the soluble extract of rat-liver mitochondria. This enzyme could use NADP in place of NAD but was not identical with liver alcohol dehydrogenase (EC 1.1.1.1). 6. [26-¹⁴C]Cholesteryl 3β-sulphate was not oxidized by fortified mouse-liver preparations that oxidized [26-¹⁴C]cholesterol to ¹⁴CO₂.     

ELECTRON MICROSCOPE HISTOCHEMICAL EVIDENCE FOR A PARTIAL OR TOTAL BLOCK OF THE TRICARBOXYLIC ACID CYCLE IN THE MITOCHONDRIA OF PRESYNAPTIC AXON TERMINALS

Respiration-linked, massive accumulation of Sr²⁺ is used to reveal the coupled oxidation of pyruvate, α-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no α-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of α-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr²⁺ accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO₂ into some of the pyruvate.     

Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [³⁵S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [³⁵S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-³H]MeSH and [³⁵S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 10⁶- to 10⁷-fold higher concentrations.     

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Δ⁵ to Δ⁷ or to Δ^5,7. Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24α-ethyl = trans-Δ²²-24α-ethyl > trans-Δ²²-24β-ethyl = 24α-E-ethylidene = 24α-methyl > 24β-methyl = trans-Δ²²-24β-methyl = 26-methyl = saturated C₇ side chain and C-20 R (17-αH, 20-αH, right-handed conformer) = cis-Δ²²-C₇ side chain and C-20 R > saturated C₇ side chain and C-20 S (17-αH, 20-βH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20α-hydroxycholesterol = 24ξ-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20α-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.