Alanine Racemase Activity Research Articles

Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase

We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL 21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 310 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K(m) and V(max) values were determined as follows: l-alanine to d-alanine, K(m) (l-alanine) 5.01 mM and V(max) 306 U/mg; d-alanine to l-alanine, K(m) (d-alanine) 5.24 mM and V(max) 345 U/mg. The K(eq) value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C. glutamicum ATCC 13032 is expected to be a biocatalyst for d-alanine production from the l-counter part.

The occurrence of free d-alanine and an alanine racemase activity inLeishmania amazonensis

Free D-amino acids are implicated in several biological functions. This study examined the presence of D-alanine in Leishmania amazonensis. Measuring chiral amino acid content by high-performance liquid chromatography we detected a significant amount of free D-alanine in promastigotes of these parasites. D-alanine accounts for 8.9% of total free alanine and is found primarily in the soluble fraction. Specific racemization of L-alanine to D-alanine was detected in cell lysates and this enzyme activity was inhibited by D-cycloserine, an alanine racemase inhibitor. Furthermore, we were able to decrease this pool of D-amino acid by treating our cultures with D-cycloserine. We demonstrate for the first time the existence of a significant amount of free D-alanine in L. amazonensis and an alanine racemase activity present in cell lysates. The restriction of D-alanine to bacteria, some fungi and now in L. amazonensis opens a new perspective on treatment of diseases caused by these microorganisms.

Purification and some properties of alanine racemase from the marine gastropod Cellana grata

High levels of free D-alanine were found in the muscle of a marine gastropod Cellana grata that inhabited the intertidal zone, and alanine racemase activity was detected in the muscle. The authors purified alanine racemase from the muscle of C. grata to characterize its enzymological properties. The molecular mass of the enzyme was estimated to be 40.5 kDa by sodium dodecylsulfatepolyacrylamide gel electrophoresis and 41.4 kDa by gel filtration, suggesting that the enzyme was monomeric in nature. Kinetic experiments, performed using the purified enzyme, revealed that the Lineweaver-Burk plot for D-alanine as a substrate resulted in a K m value of 20.4 mM, and the value for L-alanine was 43.0 mM. Of the several types of amino acids tested, alanine was found to be the specific substrate for the enzyme. In the measurement of alanine racemase activity, exogenous pyridoxal 5′-phosphate (PLP) was not required for the enzyme activity; however, aminooxyacetic acid, hydroxylamine and phenylhydrazine, which inhibit PLP-dependent enzymes, strongly inhibited the enzyme activity. These results suggest that the enzyme is PLP-dependent. This is the first report on the purification and some properties of alanine racemase in a marine gastropod.

Alanine racemase activity in the microalga Thalassiosira sp.

In this paper, the authors report the detection of alanine racemase activity in the marine diatom Thalassiosira sp. Since the Thalassiosira sp. was cultured under germ-free conditions, it appeared that D-alanine was not derived from bacteria but was produced through catalysis by algal alanine racemase. The rate of conversion of L-alanine to D-alanine was approximately the same as that for the reverse reaction, and the enzyme catalyzed the equilibration of the D- and L-forms. The crude enzyme preparation obtained from the cells at the stationary phase of the growth cycle had an optimal pH of approximately 9.5. The Lineweaver—Burk analysis showed that the K m for D- and L-alanine was 16.5 mM and 29.4 mM, respectively. It appears that the enzyme is highly specific for D- or L-alanine because it does not catalyze the racemization of other amino acids. In addition, after gel filtration, the enzyme did not require exogenous pyridoxal 5′-phosphate (PLP) for its activity, however, the effects of several chemicals suggest that the enzyme may be PLP-dependent. The enzyme is more similar to that found in invertebrates when compared with that found in bacteria. This is the first report on the occurrence of alanine racemase activity in the microalga Thalassiosira sp.

Structural Evidence That Alanine Racemase from a d-Cycloserine-producing Microorganism Exhibits Resistance to Its Own Product

Alanine racemase (ALR), an enzyme that catalyzes the interconversion of Ala enantiomers, is essential for the synthesis of the bacterial cell wall. We have shown that it is harder to inhibit the catalytic activity of ALR from D-cycloserine (DCS)-producing Streptomyces lavendulae than that from Escherichia coli by DCS. To obtain structural evidence for the fact that Streptomyces ALR displays resistance to DCS, we determined the precise nature of the x-ray crystal structures of the cycloserine-free and cycloserine enantiomer-bound forms of Streptomyces ALR at high resolutions. Streptomyces ALR takes a dimer structure, which is formed by interactions between the N-terminal domain of one monomer with the C-terminal domain of its partner. Each of the two active sites of ALR, which is generated as a result of the formation of the dimer structure, is composed of pyridoxal 5'-phosphate (PLP), the PLP-binding residue Lys(38), and the amino acids in the immediate environment of the pyridoxal cofactor. The current model suggests that each active site of Streptomyces ALR maintains a larger space and takes a more rigid conformation than that of Bacillus stearothermophilus ALR determined previously. Furthermore, we show that Streptomyces ALR results in a slow conversion to a final form of a pyridoxal derivative arising from either isomer of cycloserine, which inhibits the catalytic activity noncompetitively. In fact, the slow conversion is confirmed by the fact that each enzyme bound cycloserine derivative, which is bound to PLP, takes an asymmetric structure.

Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications.

To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, k (cat) and K (m), for L-alanine are 1.05+/-0.03 s(-1) and 10+/-1 mM respectively, whereas those for D-alanine are 1.4+/-0.1 s(-1) and 10+/-1 mM. During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP). The catalytic efficiency of the half-transamination of L-alanine is found to be 5.3x10(-5) mM(-1) x s(-1), 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3x10(3) for L-alanine and 1.4x10(4) for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with p K values of 6.5-6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4' S)- and (4' R)-[4'-(3)H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.

Characterization of an alanine racemase gene from Lactobacillus reuteri.

An alanine racemase gene from Lb. reuteri was cloned by using degenerate oligonucleotides corresponding to conserved regions derived from several bacterial alanine racemases. The protein is 375alphaalpha in length and shows 63.6% homology to the Lb. plantarum alanine racemase. Unlike the single alanine racemase activity found in Lb. plantarum, deletion of the Lb. reuteri alanine racemase reveals a second activity, which is inhibited by beta-chloro- D-alanine.

High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5′-phosphate

We examined the effect of the pyridoxal 5′-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40°C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40°C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60°C.

High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate.

We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.

Purification and properties of alanine racemase from crayfish Procambarus clarkii

Fresh water crayfish Procambarus clarkii is known to accumulate d-alanine remarkably in muscle after seawater acclimation, accompanied by an increase in alanine racemase activity. We have purified alanine racemase from crayfish muscle to homogeneity. The enzyme is a monomeric protein with a molecular mass of 58 kDa. It is highly specific to alanine and does not racemize l-serine, l-aspartate, l-glutamate, l-valine and l-arginine. The enzyme shows the highest activity at pH 9.0 in the conversion of l- to d-alanine and at pH 8.5 in the reverse conversion. Properties such as amino acid sequence, quaternary structure, pyridoxal 5′-phosphate (PLP)-dependency, pH-dependency and kinetic parameters seem to be distinct from those of the microbial alanine racemases. Various salts including NaCl at concentrations around seawater level were potently inhibitory for the activity in both of l- to - d and d- to - l direction.

Effect of ethyl alcohol on growth and intracellular alanine racemase of psychrotrophs

The psychrotrophic alanine racemase from Pseudomonas fluorescens, a typical psychrotroph, is less resistant to organic solvents than the enzymes from thermophilic and mesophilic bacteria (Okubo et al., J. Home Econ. Jpn., 46: 1135–1140, 1995). To further elucidate this difference, we examined the effect of ethyl alcohol on the growth and intracellular alanine racemase activity of three typical psychrotrophs— P. fluorescens, Bacillus psychrosaccharolyticus and B. psychrophilus—in comparison with two mesophiles, Escherichia coli and B. subtilis. Although all the bacteria grew to the early stationary phase when cultivated at 22°C for 36 h in the absence of ethyl alcohol, the growth of the psychrotrophs was more effectively suppressed by the addition of 3 and 5% ethyl alcohol to the medium than that of the mesophiles. The intracellular alanine racemase activity of the psychrotrophs was also more markedly reduced in the presence of ethyl alcohol than that of the mesophiles. When bacterial cells of each strain grown at 22°C for 36 h in the absence of alcohol were suspended in 0–5 % ethyl alcohol solution and incubated at 30°C for 1 h, both the survival ratio and intracellular alanine racemase activity of the psychrotrophs were lower than those of the mesophiles. Thus, ethyl alcohol effectively reduced both the growth of the psychrotrophs and their intracellular alanine racemase activity. Low concentrations of various other alcohols also repressed the growth of the psychrotrophs at 10°C.

The alanine racemase gene is essential for growth of Lactobacillus plantarum.

The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr- DadX- double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.

Occurrence of Alanine Racemase in Crustaceans and the Changes of the Properties During Seawater Acclimation of Crayfish

Alanine racemase activity was detected in the muscle and hepatopancreas of six macruran species. Optimal pH was around 8.5 irrespective of species and tissues. Apparent Michaelis constants ranged from 48 to 157 mM for muscle enzyme and 35 to 239 mM for hepatopancreas enzyme. The enzyme specifically catalyzed the racemization of d- and l-alanine. The enzyme did not require pyridoxal 5′-phosphate as well as FAD as a cofactor. Pyruvate and l-alanine showed strong inhibition in the direction of d to l. During seawater acclimation of crayfish Procambarus clarkii, alanine racemase activity increased about twice in muscle and 1.5 times in hepatopancreas. Michaelis constant, on the other hand, decreased 33% for muscle enzyme and 65% for hepatopancreas enzyme, suggesting the increase of substrate affinity during seawater acclimation. The activity in the physiological pH range (6.5–7.5) also increased with increasing salinity.

Different physiological functions of free D- and L-alanine in three body parts of the intertidal sipunculid Phascolosoma arcuatum

Free D- and L-alanine contents were comparable in the body wall and introvert cum retractor muscles of Phascolosoma arcuatum. In contrast, the content of free D-alanine in the internal organs was twice that of free L-alanine. Since alanine aminotrans-ferase from P. arcuatum was L-alanine specific, D-alanine appeared to be synthesized from L-alanine through the action of alanine racemase. Alanine racemase activity was higher in the D-alanine-forming direction in the three body parts of P. arcuatum. In addition, the ratio of D→L/L→D racemase activity in the internal organs was the lowest among the body parts studied. These results indicate that free D-alanine might be of lesser importance than the free D-isomer to the internal organs as compared to the body wall and introvert cum retractor muscles. Indeed, L-alanine inhibited pyruvate kinase from the body wall and introvert cum retractor muscles but had no effect on the pyruvate kinase from the internal organs. Furthermore, the activity of alanopine dehydrogenase present in the internal organs was significantly lower than those of the body wall and introvert cum retractor muscles. L-Alanine was an essential substrate for alanopine formation in the body wall and introvert cum retractor muscles during hypoxia since alanopine dehydrogenases from these body parts were L-alanine specific. When P. arcuatum was confronted with hypo-osmotic stress, the free D-alanine/total free alanine ratio in the internal organs increased approximately from 0.6 to 0.8 as the total free alanine content decreased. In comparison, those ratios in the body wall and introvert cum retractor muscles remained relatively constant. It was concluded that D- and D-alanine had different physiological functions in the three body parts of P. arcuatum.

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L → D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.

The relation of d-alanine and alanine racemase activity in molluscs

1. 1. Eighteen molluscan species were examined for the presence of d-alanine and alanine racemase activity to probe the probable relation between them. 2. 2. Two bivalve species had high concentration of d-alanine and l-alanine (1:1) and showed high activities of alanine racemase. In these species, the occurrence of d-alanine could be explained by the action of alanine racemase. 3. 3. In other species, the levels of d-alanine and enzyme activity were low, and the occurrence of d-alanine did not correspond with the presence of alanine racemase activity. 4. 4. The mechanism of the occurrence of d-alanine in molluscan tissues seems to vary from species to species and seems not to be associated with the phylogenic situation or habitats of the respective species.

Peptidoglycan biosynthesis in Escherichia coli: variations in the metabolism of alanine and D-alanyl-D-alanine.

The in vivo functioning of the alanine/D-alanyl-D-alanine pathway of Escherichia coli was investigated by determining precursor pool levels and specific enzyme activities under various growth conditions. Cells grown on D- or L-alanine showed several remarkable features compared with cells grown on other carbon sources: 10-fold higher values of the D-alanyl-D-alanine and the UDP-MurNAc-pentapeptide pools, a 240-fold increase of the alanine racemase activity, and the absence of bacteriolysis after treatment with D-cycloserine at high concentrations (50 micrograms ml-1). In cells grown on glucose, D-cycloserine (1 micrograms ml-1) led to depletion of the D-alanyl-D-alanine pool and to lysis, which was efficiently antagonized by chloramphenicol. A threefold increase of the dipeptide pool was observed when cells were treated with chloramphenicol alone. The alanine racemase activity was lowest in glucose-grown cells and the D-alanine:D-alanine ligase and D-alanyl-D-alanine-adding activities were the same whatever the carbon source. Molecular masses of 53-56 kDa and 56-60 kDa were estimated for the partially purified inducible alanine racemase and D-alanine:D-alanine ligase respectively.

Temperature-sensitive murein synthesis in an Escherichia coli pdx mutant and the role of alanine racemase.

The basis for disruption of morphogenesis by depletion of pyridoxine derivatives was studied using a pdxH null mutant of Escherichia coli K-12. Removal of pyridoxal from growing cultures severely inhibited murein synthesis in vivo, whereas simultaneous supplementation with D-alanine effectively prevented inhibition. Extractable alanine racemase was low following such starvation. Selection of mutants overcoming the glycine- or temperature-sensitivity imposed by pyridoxine limitation yielded a variety of phenotypes. The most effective of these extragenic suppressors conferred an elevated alanine racemase activity which was resistant to the effects of pyridoxal removal.

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization.

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.