Studies on the enzymic degradation of L-serine O-sulphate by a rat liver preparation

1. The enzyme system of rat liver responsible for the degradation of l-serine O-sulphate was purified 300-fold and the optimum conditions for the activity were determined. 2. Inorganic sulphate, pyruvate and ammonia were found to be the products of enzyme action on lserine O-sulphate, being formed in equivalent amounts under all conditions examined. No free l-serine was detected as a product of enzyme action. 3. The enzyme preparation was free from other serine-metabolizing systems such as O-phospho-l-serine phosphatase and l-serine dehydratase. 4. The enzyme has a very narrow substrate specificity and is inactive towards a wide variety of related sulphate esters and amino acids. 5. Pyridoxal 5'-phosphate is capable of catalysing the non-enzymic breakdown of l-serine O-sulphate in the presence of metal salts to yield inorganic sulphate, pyruvate and ammonia as products. 6. The possible role of pyridoxal 5'-phosphate as a coenzyme in the enzymic degradation of l-serine O-sulphate is discussed.     

Physiological comparison of L-serine dehydratase and tryptophanase from Bacillus alvei

Tryptophanase from Bacillus alvei also possesses serine dehydratase activity. A comparison of this enzyme with l-serine dehydratase [l-serine hydro-lyase (deaminating), EC 4.2.1.13] in toluene-treated whole cell preparations of the organism was undertaken. Tryptophanase is a constitutive enzyme in B. alvei. The dehydratase undergoes a repression-derepression-repression sequence as the l-serine level in the growth medium is increased from 0 to 0.1 m. Tryptophanase activity is decreased in organisms grown in medium containing glucose. Both enzymes are repressed in organisms grown in glycerol-containing medium. l-Serine dehydratase has a pH optimum of 7.5 in potassium phosphate buffer; tryptophanase functions optimally in this buffer at pH 8.2. Both enzymes lose activity in the presence of tris(hydroxymethyl)aminomethane buffer. Either K(+) or NH(4) (+) is required for full tryptophanase activity, but Na(+) is markedly inhibitory. These three cations are stimulatory to l-serine dehydratase activity. Both enzymes are subject to apparent substrate inhibition at high concentrations of their respective amino acids, but the inhibition of tryptophanase activity can be completely overcome by the removal of indole as it is formed. The dehydratase does not catalyze cleavage of d-serine, l-threonine, or alpha-substituted serine analogues at the concentrations tested. However, activity of the enzyme in cleaving l-serine is competitively inhibited by d-serine, indicating that the d-isomer can occupy an active site on the enzyme. The enzyme catalyzes cleavage of some beta-substituted serine analogues.     

Allosteric regulation of mouse brain serine racemase

Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg²⁺ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for l-serine decreased from 13 mM to 1.8 mM with little change in V _max, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on l-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. l-serine was also converted to pyruvate at about twice the racemization rate. l-serine O-sulfate dehydration was inhibited by ATP, while l-serine dehydration, like racemization, was activated by nucleotides, indicating that, for l-serine, dehydration and racemization take place at the same site.     

Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition

Without significant killing, d-serine at concentrations greater than 50 mug/ml inhibits growth in minimal media of mutants of Escherichia coli K-12 unable to form d-serine deaminase. The mutants eventually recover at lower concentrations. There is no evidence of d-serine toxicity in rich media. Toxicity is partially reversed by l-serine. d-Serine does not interfere with l-serine activation, one-carbon metabolism, or (Cronan, personal communication) formation of phosphatidylserine. Pizer (personal communication) finds, however, that it is a powerful feedback inhibitor of the first enzyme of l-serine biosynthesis. In the presence of l-serine, the residual toxicity is largely and noncompetitively over come by pantothenate, indicating that d-serine inhibits growth by affecting two targets: pantothenate biosynthesis and l-serine biosynthesis. l-Serine causes transient growth inhibition in E. coli K-12. Contaminating l-serine in d-serine preparations contributes to the d-serine inhibitory response.     

Removal of the tryptophan 139 side chain in Escherichia coli D-3-phosphoglycerate dehydrogenase produces a dimeric enzyme without cooperative effects

Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) is a homotetrameric enzyme whose activity is allosterically regulated by l-serine, the end-product of its metabolic pathway. Previous studies have shown that PGDH displays two modes of cooperative interaction. One is between the l-serine binding sites and the other is between the l-serine binding sites and the active sites. Tryptophan 139 participates in an intersubunit contact near the active site catalytic residues. Site-specific mutagenesis of tryptophan 139 to glycine results in the dissociation of the tetramer to a pair of dimers and in the loss of cooperativity in serine binding and between serine binding and inhibition. The results suggest that the magnitude of inhibition of activity at a particular active site is primarily dependent on serine binding to that subunit but that activity can be modulated in a cooperative manner by interaction with adjacent subunits. The disruption of the nucleotide domain interface in PGDH by mutating Trp-139 suggests the potential for a critical role of this interface in the cooperative allosteric processes in the native tetrameric enzyme.     

Kinetic Evidence of a Noncatalytic Substrate Binding Site That Regulates Activity in Legionella pneumophilal-Serine Dehydratase

The l-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (β domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial l-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, l-serine binds to the ACT domain to inhibit catalytic activity. However, substrate must be bound to the ASB domain for serine to exert its effect. As such, the ASB domain acts as a codomain for the action of l-serine. Pre-steady-state kinetic analysis of l-serine binding to lpLSD demonstrates that l-serine binds to a second noncatalytic site and produces a conformational change in the enzyme. The rate of this conformational change is too slow for its participation in the catalytic cycle but rather occurs prior to catalysis to produce an activated form of the enzyme. That the conformational change must occur prior to catalysis is shown by a lag in the production of product that exhibits essentially the same rate constant as the conformational change. The second, noncatalytic site for l-serine is likely to be the ASB domain (β domain) of lpLSD that functions in a manner similar to that in PGDH. A mechanism whose overall effect is to keep l-serine levels from accumulating to high levels while not completely depleting the l-serine pool in the bacterial cell is proposed.     

Enzymatic methods for the determination of L-serine concentration and L-[14C]serine specific radioactivity in blood plasma

Methods are desribed for the use of l-serine dehydratase purified from Clostridium acidiurici for the determination of l-serine concentration and l[¹⁴C]serine specific radioactivity in sheep plasma. A spectrophotometric assay using this enzyme accurately measured the concentration of l-serine in standard solutions and in a commercially available mixture of amino acids and related compounds. This assay was shown to be suitable for measurement of plasma l-serine concentrations in excess of 30 μm. The reverse isotope dilution method was used for plasma l-[¹⁴C]serine specific radioactivity measurements. Carrier l-serine was added to plasma and separated from neutral and anionic compounds using ion-exchange chromatography. The l-serine was then converted to pyruvate with l-serine dehydratase and this was purified as the phenylhydrazone derivative. After recrystallization, drying and weighing, the derivative was assayed for radioactivity. The accuracy of this method was verified by adding l-[U-¹⁴C]serine to plasma and comparing the experimentally determined l-[¹⁴C]serine specific radioactivity with the calculated value. The method yielded a value which was 98.6 ± 0.8% (5) of this calculated value.     

Effects of l-serine dehydratase activity on l-serine production by Corynebacterium glycinophilum and an examination of the properties of the enzyme

The results with Corynebacterium glycinophilum AJ-3170 and various mutants from AJ-3170 indicated that l-serine production was almost inversely proportional to l-serine degrading activity. The crude extract of the parental strain, AJ-3170, showed l-serine and l-threonine degrading activities. The 2 activities were completely separated from each other by gel-filtration, indicating that each activity comes from a different enzyme. The l-serine degrading enzyme, l-serine dehydratase (SD), was purified 30-fold from AJ-3170. Molecular weight of SD was 130,000. The enzyme was specific for l-serine, activated slightly by FeCl₂ and inhibited by MnCl₂. The double reciprocal plots of SD rate against substrate concentration gave an upwards-curved line. The value of [S]_0.5 was 35 mM.     

Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Production of l-serine from glycine by Corynebacterium glycinophilum and properties of serine hydroxymethyltransferase,a key enzyme in l-serine production

Fermentative production of l-serine from glycine by Corynebacterium glycinophilum AJ-3413, an auxotrophic mutant of Leu and Met with increased productivity of l-serine using a one liter jar fermentor was carried out and the properties of serine hydroxymethyltransferase (SHMT), a key enzyme in l-serine synthesis, of the parental strain AJ-3170 were investigated. SHMT was effectively induced by the addition of glycine to the medium at an early stage of cultivation. Under optimal conditions, AJ-3413 produced 16.0 g/l of l-serine from 30 g/l of glycine with a molar yield of 38%. The partially purified SHMT catalyzed the l-allo-threonine degradation in addition to l-serine degradation, but could not catalyze l-threonine degradation. This enzyme showed an absolute tetrahydrofolic acid requirement for l-serine degradation to glycine and formaldehyde, but not for l-allo-threonine degradation. Pyridoxal 5′-phosphate appeared to be required for enzyme activity. The K_m values for glycine and formaldehyde in l-serine synthesis, and for l-serine in l-serine degradation were 1.85, 0.29 and 1.64 mM, respectively.     

Allosteric Activation and Contrasting Properties of l-Serine Dehydratase Types 1 and 2

Bacterial l-serine dehydratases differ from mammalian l- and d-serine dehydratases and bacterial d-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 d-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between l-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with l-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of l-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.     

A new enzymatic reaction for producing new-type amino acids by Escherichia coli: Production of α-amino-β-(1-indoline) propionic acid from indoline and l-Serine

We found that some reaction products were produced from indole-mimic compounds, such as indoline (2,3-dihydroindole), indazole, 7-azaindole and 3-indazolinone, with l-serine by the catalytic action of the lyophilized cells of Escherichia coli T4-3 (a mutant defective in indole-3-glycerolphosphate synthase [EC 4.1.1.48]) cultured in a tryptophan-limited medium.A main product from indoline and l-serine was isolated and identified as a-amino-β-(1-indoline) propionic acid (AIP) from data obtained by paperchromatography, elemental analysis, UV, IR, ¹H-NMR and mass spectrometry.The reaction conditions and the requirements for the reaction were also studied.AIP was produced only in the case of using l-serine, l-serine methylester and l-serine ethylester as the amino acid source.On the enzyme concerned AIP production, studies were carried out by using the mutant strains of E. coli defective in the enzyme(s) of tryptophan operon. Tryptophan synthase [EC 4.2.1.20], particularly its B protein, was presumed to be a possible candidate.     

Mechanistic studies of p-hydroxybenzoate hydroxylase reconstituted with 2-Thio-FAD

2-Thio-FAD (oxygen substituent at position 2 is replaced by sulfur) was used to reconstitute the apoenzyme of p-hydroxybenzoate hydroxylase. The 2-thio-FAD enzyme differs from native enzyme in several respects. While the native enzyme catalyzes the fully coupled hydroxylation of p-hydroxybenzoate, the 2-thio-FAD enzyme shows no hydroxylation of this substrate, instead reducing molecular oxygen to hydrogen peroxide. The rate of reduction of 2-thio-FAD p-hydroxybenzoate hydroxylase by NADPH in the presence of substrate was 7-fold faster than with the native enzyme. However, the oxygen reactivity of the reduced 2-thio-FAD enzyme was less than 1% that of native enzyme. This slow oxygen reaction results in the very high KmO2 observed in steady state kinetic studies of the modified enzyme. Stopped flow studies of the oxygen reaction of the reduced 2-thio-FAD enzyme in the presence of substrate confirmed the formation of a transient intermediate. The spectrum of this intermediate is very similar to those of the flavin-C(4a) adducts obtained with 2-thio-FMN lactate oxidase. This evidence suggests that reduced 2-thio-FAD p-hydroxybenzoate hydroxylase forms a flavin-C(4a)-hydroperoxide on reaction with oxygen in a reaction analogous to that with native enzyme, but that the resulting peroxyflavin is incompetent as an oxygenating species, breaking down instead to oxidized 2-thio-FAD enzyme and hydrogen peroxide.     

Increased liver l-serine–pyruvate aminotransferase activity under gluconeogenic conditions (Short Communication)

Rat liver l-serine-pyruvate aminotransferase activity exceeds markedly the normal adult value (a) in the neonatal period, (b) after glucagon injection and (c) after alloxan injection, observations that reinforce the suggestion from comparative findings that the aminotransferase has a role in gluconeogenesis. Some findings, however, argue in favour of l-serine dehydratase as the enzyme of gluconeogenesis from l-serine.     

Acceleration of the substrate Calpha deprotonation by an analogue of the second substrate palmitoyl-CoA in Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.     

Functional properties of the active core of human cystathionine beta-synthase crystals

Human cystathionine beta-synthase is a pyridoxal 5'-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate l-serine readily led to the formation of the key catalytic intermediate alpha-aminoacrylate. The dissociation constant of l-serine was found to be 6 mm, close to that determined in solution. The amount of the alpha-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the alpha-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the alpha-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta-synthase crystals catalyze both beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta-synthase.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction

Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity and purified SPT to homogeneity. This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2. The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5'-phosphate-dependent enzyme. The substrate specificity of the Sphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT shows about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme. This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.     

Growth Inhibition of Streptomyces Species by l-Serine and Its Effect on Tetracycline Biosynthesis

The addition of serine to minimal medium inhibited the growth of Streptomyces aureofaciens and Streptomyces rimosus. Both the outgrowth of spores and the growth of vegetative cells were inhibited by l-serine. This effect was independent of the carbon source used. In rich nutrient medium, however, the serine effect was not observed. The presence of glycine and methionine in minimal medium reversed the growth inhibition imposed by serine, suggesting that a metabolic block related to the synthesis of these two amino acids was involved. A serine-tolerant mutant of S. aureofaciens isolated after ultraviolet irradiation showed a level of serine deaminase comparable to that of the wild-type strain, which indicated that tolerance to serine was not due to the presence of a more active deaminating enzyme in the mutant. Serine markedly reduced tetracycline and oxytetracycline biosynthesis with the parental strains of Streptomyces spp. The serine-tolerant mutant, however, produced almost the same amount of tetracycline in the presence or absence of serine. The final cell population in fermentation broth was not significantly reduced by l-serine, and the addition of glycine and methionine did not increase the tetracycline yields, which suggested that l-serine inhibition of antibiotic biosynthesis was by a mechanism different from that related to growth inhibition.     

Structural basis for acyl acceptor specificity in the achromobactin biosynthetic enzyme AcsD

Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential.