Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes

We derived l-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selected the potent l-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg l-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The l-methionine biosynthetic enzymes, cystathionine γ-synthase and cystathionine β-lyase, of the l-methionine-overproducing mutants were little repressed by l-methionine. To analyse the mechanism of l-methionine overproduction in the mutant strains, the metJ gene coding for the E. colimet repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser → Asn) at position 54 was observed in four independent l-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the l-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the l-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in l-methionine overproduction, probably because of the derepression of l-methionine biosynthetic enzymes. Received: 6 January 1999 / Received last revision: 19 February 1999 / Accepted: 26 February 1999     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

d-Methionine preparation from racemic methionines by Proteus vulgaris IAM 12003 with asymmetric degrading activity

The microbial degradation of l-methionine was investigated in order to develop a practical process for d-methionine production from racemic methionines. Among the 1000 culture strains tested, microorganisms belonging to the Achromobacter, Bacillus, Micrococcus, Morganella, Proteus, Providencia, Pseudomonas and Sarcina genera exhibited a high l-methionine-degrading activity. Proteus vulgaris IAM 12003 was determined to be the best strain and was used as a biocatalyst for eliminating the l-isomer. The degradation of l-isomer in this P. vulgaris IAM 12003 cell was assured by the action of l-amino acid oxidase. The maximum rate of l-isomer degradation was obtained at 30 °C and pH 8.0. Under these optimal conditions, the l-isomer in a 100 g/l mixture of racemic methionines was almost degraded within 20 h, with 46.5 g d-methionine/l remaining in the reaction mixture. Crystalline d-methionine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 30% from the reaction mixture by simple purification. Received: 17 June 1996 / Received last revision: 11 September 1996 / Accepted: 29 September 1996     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Enantioselective synthesis ofN-acetyl-l-methionine with aminoacylase in organic solvent

We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl₂, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids.     

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its α,γ-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH₃SeH from seleno-l-methionine (l-SeMet) by the α,γ-elimination reaction. The l-SeMet α,γ-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The K _m value of the enzyme for the catalysis of l-SeMet was 15.5 m M, and the V _max was 0.29 units/mg protein. Pyridoxal 5′-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50°C; it catalyzed the α,γ-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the α,β-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inerts. Therefore, the purified enzyme was different from the bacterial l-methionine γ-lyase that metabolizes l-SeMet to CH₃SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the α,γ-elimination reaction of l-SeMet and immediately converts it to CH₃SeH, an important metabolite of Se.     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Comparison of volatile sulphur compound production by cheese-ripening yeasts from methionine and methionine–cysteine mixtures

Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with l-methionine or l-methionine/l-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with l-methionine or l-methionine/l-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when l-cysteine was supplemented, even at a low concentration (0.2 g l⁻¹). This effect was due mainly to a significant decrease in l-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by l-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

Inhibition of thermolysin by 3-trimethylsilylalanine derivatives

The inhibition of thermolysin by an optically active silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), and its derivatives was examined by considering the similarity of structure between TMS-Ala and leucine. Although free l- and d-TMS-Ala did not show the inhibition, several derivatives of l-TMS-Ala, especially Z-l-TMS-Ala and l-Leu-(l-TMS-Ala), exhibited a higher inhibitory activity toward thermolysin than did Z-l-Leu and l-Leu-l-Leu respectively. Effects of TMS-Ala on the activity of its derivatives and the mode of interaction between the derivatives of TMS-Ala and thermolysin are also discussed. Received: 24 February 1999 / Received last revision: 4 June 1999 / Accepted: 27 June 1999     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

<Emphasis Type="SmallCaps">l</Emphasis>-Methioninase Production by Filamentous Fungi: I-Screening and Optimization Under Submerged Conditions

Findings show 21 fungal isolates belonging to eight genera recovered from Egyptian soils that have the potential to attack l-methionine under submerged conditions. Aspergillus flavipes had the most methioninolytic activity, giving the highest yield of l-methioninase (10.78 U/mg protein), rate of methionine uptake (93.0%), and growth rate (5.0 g/l), followed by Scopulariopsis brevicaulis and A. carneus. The maximum l-methioninase productivity (11.60 U/mg protein) by A. flavipes was observed using l-methionine (0.8%) as an enzyme-inductive agent and glucose (1%) as a co-dissimilated carbon source. A significant reduction in l-methioninase biosynthesis by A. flavipes was detected using carbon-free medium, suggesting the lack of ability to use l-methionine as a carbon and nitrogen source. Potassium dihydrogen phosphate (0.25%), the best source of phosphorus, favors enzyme biosynthesis and enhances the level of methionine uptake by A. flavipes. The maximum l-methioninase productivity (12.58 U/mg protein) and substrate uptake (95.6%) were measured at an initial pH of 7.0.     

Isolation and Characterization of S-Adenosylmethionine-requiring Mutants and Role of S-Adenosylmethionine in the Regulation of Methionine Biosynthesis in Corynebacterium glutamicum

Using a minimal medium containing a methionine analog together with a small amount of S-adenosylmethionine (SAM), many SAM requiring mutants which responded only to SAM and not to methionine, S-adenosylhomocysteine, or homocysteine were efficiently isolated from Corynebacterium glutamicum TLD-140 after mutagenesis. Among them, SAM-14 and SAM-19 selected from selenomethionine resistant mutants were subjected to further investigation. Both mutants were unable to grow in a minimal medium and had no detectable activity of SAM synthetase. Both mutants acquired higher resistance to methionine hydroxamate and ethionine as well as to selenomethionine than TLD-140 and produced l-methionine in a medium.

Homoserine-O-transacetylase in SAM-19 was subject to full repression by the addition of excess SAM to the growth medium and was not repressed under SAM limitation, whereas addition of excess l-methionine under SAM limitation caused a partial repression of the enzyme. SAM synthetase as well as l-methionine biosynthetic enzymes in a methionine auxotroph of C. glutamicum was repressed by the addition of l-methionine to the growth medium.

These results suggest that SAM is implicated in the repression of l-methionine synthesizing enzymes in C. glutamicum.     

Catalytic effects of histidine enantiomers and glycine on the formation of dileucine and dimethionine in the salt-induced peptide formation reaction

The salt-induced peptide formation (SIPF) reaction takes place readily under mild reaction conditions and proceeds via a copper complex. Its ease of reaction and the universality for prebiotic scenarios add weights to the arguments in favour of the importance of peptide and proteins in the tug of war with the RNA world hypothesis. In addition, the SIPF reaction has a preference for l-form amino acids in dipeptide formation, casting light on the puzzle of biohomochirality, especially for the amino acids with aliphatic side chains. A detailed investigation on the behaviour of aliphatic leucine in the SIPF reaction is presented in this paper, including the catalytic effects of glycine, l- and d-histidine as well as the stereoselectivity under all the reaction conditions above. The results show a relatively low reactivity and stereoselectivity of leucine in the SIPF reaction, while both glycine and histidine enantiomers remarkably increase the yields of dileucine by factors up to 40. Moreover, a comparative study of the effectiveness of l- and d-histidine in catalysing the formation of dimethionine was also carried out and extends the scope of mutual catalysis by amino acid enantiomers in the SIPF reaction.     

Emodin O-methyltransferase from Aspergillus terreus

Emodin O-methyltransferase, an enzyme catalyzing methylation of the 8-hydroxy group of emodin, was identified in the mould Aspergillus terreus IMI 16043, a (+)-geodin producing strain. The enzyme catalyzed the formation of questin from emodin and S-adenosyl-l-methionine. By chromatography on DEAE-cellulose, Phenyl Sepharose, Q-Sepharose, Hydroxyapatite, and CM-cellulose, emodin O-methyltransferase was purified to apparent homogeneity. The purified protein had a molecular weight of 322 kDa as estimated by gel filtration and 53.6 kDa as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homohexamer. The enzyme showed pI 4.4 and optimum pH 7–8. Magnesium ion or manganese ion was not an absolute requirement, nor increased the enzyme activity. The enzyme had strict substrate specificity and very low Km values for both emodin (3.4×10^-7 M) and S-adenosyl-l-methionine (4.1×10^-6 M).Abbreviations EOMT emodin O-methyltransferase from A. terreus - SAM S-adenosyl-l-methionine - PAGE polyacrylamide gel electrophoresis     

Overexpression of yeast <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase <Emphasis Type="Italic">metK</Emphasis> in <Emphasis Type="Italic">Streptomyces actuosus</Emphasis> leads to increased production of nosiheptide

S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and l-methionine that is catalyzed by the enzyme S-adenosyl-l-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Regulation of homoserine dehydrogenase inAzotobacter species

The regulation of homoserine dehydrogenase activity was studied in nineAzotobacter strains belonging to five different species. In all the species the enzyme is subject to feedback inhibition byl-threonine andl-isoleucine, the first being much more active as inhibitor. The inhibition byl-threonine is noncompetitive with respect to NADPH and of mixed type with respect to aspartate-Β-semialdehyde; the inhibition byl-isoleucine is noncompetitive with respect to both substrates. The synthesis of homoserine dehydrogenase inAzotobacter chroococcum I.P. is somewhat repressed by 1mm l-methionine and 5mm l-isoleucine. In all the strains examined either NADPH or NADH can serve as cofactors for this activity, though the ratio of activity with the two pyridine nucleotides (NADPH/NADH) shows higher values (3.3–3.8) in the speciesmacrocytogenes andinsignis than in thechroococcum, beijerinckii andvinelandii group (1.5–1.6). The pattern of control of this enzyme in the genusAzotobacter is discussed in relation to other bacterial homoserine dehydrogenases. We are grateful to Dr. G. N. Cohen, Service de Physiologie Microbienne, Institut Pasteur, Paris, for helpful discussions and encouragements.     

Production of O-Acetyl-l-homoserme by Methionine Analogresistant Mutants and. Regulation of Homosenne-O-transacetylase in Gorynebacterium glutamicum

A large amount of O-acetyl-l-homoserine (OAH) was found to be produced by trifluo-romethionine-resistant mutants derived from Corynebacterium glutamicum ESLR–146 (Thr?,ethionine^R, selenomethionine^R) and ET_zR–606(Thr?,ethionine^R, 1,2,4-triazole^R) by mutational treatment with ethyl methanesulfonate. Some cultural conditions for OAH production were examined with one of the mutants, ESLFR-736, which was derived from ESLR–146. Addition of l-methionine or l-serine decreased OAH production. Optimal level of l-threo- nine, a growth factor in ESLFR–736, for OAH production was about 200 μg/ml, and further addition of excess l-threonine repressed OAH production. Corn steep liquor (CSL) and yeast extract added simultaneously enhanced OAH production to a great extent. Thus, the amount of OAH production reached to a level of 10.5 mg/ml with a medium containing 10% glucose and 0.01 % of both CSL and yeast extract after 2 days incubation.

Cell-free extract of C. glutamicum catalyzed the formation of OAH from acetyl CoA and l-homoserine, while a corresponding reaction with succinyl CoA was hardly detected. These observations indicate that OAH but not O-succinyl-l-homoserine is an intermediate of l-methionine biosynthesis in C. glutamicum.

The regulation of homoserine-O-transacetylase was examined in a methionine requiring mutant of C. glutamicum. The enzyme activity was not inhibited by l-methionine, S-adenosyl-methionine and S-adenosylhomocysteine, separately or in combination. The synthesis of homoserine-O-transacetylase was strongly repressed by l-methionine. The enzyme level in an OAH producer, ESLFR–736, increased to about 2-fold of that in ESLR–146, the parental strain.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996