Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity

Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.     

Structural insights of the MenD from Escherichia coli reveal ThDP affinity

MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate) synthase belongs to the superfamily of thiamin diphosphate-dependent decarboxylases, which converts isochorismate and 2-oxoglutarate to SHCHC, pyruvate, and carbon dioxide. Here, we report the first crystal structure of apo-MenD from Escherichia coli determined in tetragonal crystal form. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes. Analytical gel filtration shows that EcMenD behaves as a dimer as well as a tetramer. Circular dichroism and isothermal calorimetry results confirm EcMenD dependency on ThDP, which concomitantly helps to stabilize with better configuration.     

Specificity and reactivity in menaquinone biosynthesis: the structure of Escherichia coli MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase)

The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, or MenD, catalyze the Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate and carbon dioxide. This reaction represents the first committed step for biosynthesis of menaquinone, or vitamin K2, a key cofactor for electron transport in bacteria and a metabolite for posttranslational modification of proteins in mammals. The medium-resolution structure of MenD from Escherichia coli (EcMenD) in complex with its cofactor and Mn2+ has been determined in two related hexagonal crystal forms. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes in which two of the domains bind and force the cofactor into a configuration that supports formation of a reactive ylide. The structures reveal a stable dimer-of-dimers association in agreement with gel filtration and analytical ultracentrifugation studies and confirm the classification of MenD in the pyruvate oxidase family of ThDP-dependent enzymes. The active site, created by contributions from a pair of subunits, is highly basic with a pronounced hydrophobic patch. These features, formed by highly conserved amino acids, match well to the chemical properties of the substrates. A model of the covalent intermediate formed after reaction with the first substrate alpha-ketoglutarate and with the second substrate isochorismate positioned to accept nucleophilic attack has been prepared. This, in addition to structural and sequence comparisons with putative MenD orthologues, provides insight into the specificity and reactivity of MenD and allows a two-stage reaction mechanism to be proposed.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Steady-state kinetics and molecular evolution of Escherichia coli MenD [(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase], an anomalous thiamin diphosphate-dependent decarboxylase-carboligase

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.     

Organelle-specific Isozymes of Aspartate-alpha-Ketoglutarate Transaminase in Spinach Leaves

Four distinct isozymes of aspartate-α-ketoglutarate transaminase in a spinach (Spinacia oleracea L.) leaf extract were separated by starch gel electrophoresis. Of the total aspartate-α-ketoglutarate transaminase activity, approximately 45% was represented by the chloroplast isozyme, 26% by the cytosol isozyme, 19% by the mitochondrial isozyme, and 3 to 10% by the peroxisomal isozyme. The aspartate-α-ketoglutarate transamination activity in the four subcellular compartments behaved similarly. It was freely reversible and α-ketoglutarate was preferred to pyruvate or glyoxylate as the amino group acceptor. With glutamate as the amino group donor, oxaloacetate was superior to pyruvate or glyoxylate as the acceptor in chloroplasts, mitochondria, and cytosol, while pyruvate or glyoxylate was preferred to oxaloacetate as the acceptor in peroxisomes.     

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R)-[2(R,S)-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2(R,S)-yl-methyl)-[1,3]dioxolan-4(S)-yl]-5H-furan-2-one and 3-O-octadecyl derivatives

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of α-tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a–d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic α-tocopherol analogue (5) and natural α-tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a–d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 μM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.     

Human Regulatory Subunit RIβ of cAMP-Dependent Protein Kinases: Expression, Holoenzyme Formation and Microinjection into Living Cells

The human regulatory subunit RIβ of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S -transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant Riff protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RIβ antiserum. Furthermore, the purified recombinant RIβ protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit Cα. The rate of RIβ₂Cα₂ holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active Cα-subunit, due to cAMP binding to RIβ. Compared to a RIα₂Cα₂ holoenzyme, the RIβ₂Cα₂ holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of Riff was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RIβ was injected, whereas free Cα injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled Riff and unlabeled Cα, or unlabeled RIβ and labeled Cα, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RIβ was the labeled subunit of the in vivo dissociated bioenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled Cα from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.     

Pathway of Propionate Oxidation by a Syntrophic Culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His₆-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX₂₄TX₁₃₁HX₁₀R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent K_m values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent K_m values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

Decarboxylation of α-Keto Acids by Streptococcus lactis var. maltigenes

Decarboxylation rates for a series of C-3 to C-6 α-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for α-carboxylases. Pyruvate and α-ketobutyrate did not behave as α-carboxylase substrates, in that O₂ was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO₂ produced by some “homofermentative” streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of α-ketoisocaproate and α-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated.     

Sequential synthesis and C-N.m.r. spectra of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides

The reaction of 2,3-di-O-acetyl-4-O-benzyl-α,β-d-xylopyranosyl bromide (2) with methyl 2,3-di-O-acetyl-β-d-xylopyranoside gave methyl O-(2,3-di-O-acetyl-4-O-benzyl-β-d-xylopyranosyl)-(1→4)-2,3-di-O-acetyl-β-d-xylopyranoside (22). Catalytic hydrogenolysis of 22 exposed HO-4′ which was then condensed with 2. This sequence of reactions was repeated three more times to afford, after complete removal of protecting groups, a homologous series of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides. ¹³C-N.m.r. spectra of the synthetic methyl β-glycosides (di- to hexa-saccharide) are presented together with data for six other, variously substituted, homologous series of (1→4)-d-xylo-oligosaccharides.     

Monitoring of α-ketoglutarate in a fermentation process using expanded bed enzyme reactors

A bienzyme flow injection system is presented for the monitoring of α-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, α-ketoglutarate was converted to -glutamate by GDH in the presence of ammonia and NADH. In the following reactor, -glutamate was converted by GlOx to α-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of α-ketoglutarate in the coupled packed bed reactors was 1 μM (defined as 3 S/N), the linear range 0–100 μM, and the sensitivity 0.80 nA/μM (R² 0.99). In the coupled expanded bed reactors, the detection limit of α-ketoglutarate was 7 μM (defined as 3 S/N), the linear range and the sensitivity being 0–500 μM and 0.11 nA/μM (R² 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

New Δ8(14)-15-Ketosterols with an Oxygenated Side Chain

New analogues of 3β-hydroxy-5α-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3β-hydroxy-24-methyl-22,23-oxido-5α -cholest-8(14)-en-15- ones and (22RS,23ξ,24S)-24-methyl-5α-cholesta-8(14)-ene-3β, 22,23-triol-15-one] were synthesized from (22E,24S)-3β-acetoxy-24-methyl-5α-cholesta-8(14), 22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC₅₀ 0.9±0.2 and 0.7±0.2 μM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC₅₀ 4.0±0.5 μM), (22E,24S)-3β-hydroxy-24-methyl-5α-cholesta-8(14),22- dien-15-one (IC₅₀ 3.1±0.4 μM), and the 3β,22,23-triol synthesized (IC₅₀ 6.0±1.0 μM).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 312–319.Original Russian Text Copyright © 2005 by Flegentov, Piir, Medvedeva, Tkachev, Timofeev, Misharin.     

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [¹⁴C]Arg, [¹⁴C]Orn, and [¹⁴C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [¹⁴C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.     

Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an α-keto acid such as α-ketoglutarate (α-KG) as the amino group acceptor, and produced α-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces α-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without α-KG addition. In the presence of α-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced α-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from α-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an α-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the α-keto acid conversion to acids in L. helveticus and S. thermophilus is an α-keto acid dehydrogenase that produces acyl coenzymes A.     

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

The first step of amino acid degradation in lactococci is a transamination, which requires an α-keto acid as the amino group acceptor. We have previously shown that the level of available α-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding α-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce α-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added α-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added α-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding α-ketoglutarate to improve aroma development in cheese.     

Vitamin K (menaquinone) biosynthesis in bacteria: purification and probable structure of an intermediate prior to o-succinylbenzoate

The first aromatic intermediate in the menaquinone biosynthetic pathway is o-succinylbenzoate (OSB); it is formed from chorismate/isochorismate and 2-ketoglutarate. Cell-free extracts of menD+ E. coli strains synthesize an intermediate, "X", which is converted to OSB by extracts of menC+ cells. "X" has been purified to near homogeneity by HPLC. On treatment with acid, it yields both OSB and succinylbenzene (SB). This and other data, suggest that "X" has the structure, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (I).     

Emollient, humectant, and fluorescent alpha,beta-unsaturated thiol esters for long-acting skin applications

We describe compounds in which an emollient or a humectant bears an α,β-unsaturated thiol ester capable of reacting with nucleophilic amino acids in stratum corneum proteins. These compounds should serve as long-lasting moisturizers for skin. The emollient derivatized was octadecyl propanoate, and the humectant was poly(ethylene glycol). These hydrophobic and hydrophilic compounds, as well as a fluorescent, dansyl-containing thiol ester, were found to react within minutes with the thiol N-acetylcysteamine upon addition of a catalytic amount of an organic base in chloroform. The structures of the products resulting from conjugate addition to the unsaturated thiol esters were determined by NMR spectroscopy. In the case of the α,β,γ,δ-unsaturated (sorboyl) thiol ester, both the 1,4-addition product and the β,γ-unsaturated-1,6-addition product formed, followed by diadduct. An in vivo test of the fluorescent α,β-unsaturated thiol ester showed that this compound persisted on skin for 3 weeks vs. 6 days for the non-bonding control compound.