Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Alanine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources
PubMed1970

Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine, and glycine.

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10(-5)m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect.

Alanine↗
PubMed1975

Coupling of alanine racemase and D-alanine dehydrogenase to active transport of amino acids in Escherichia coli B membrane vesicles.

Isolated membrane vesicles from Escherichia coli B grown on DL-alanine-glycerol carry out amino acid active transport coupled to D-alanine oxidation by a membrane-bound dehydrogenase. Several other D-amino acids are substrates for this D-alanine dehydrogenase and also drive concentrative uptake of solutes. Additionally, L-alanine and L-serine can energize solute transport by virtue of conversion to oxidizable D isomers by a membrane-bound alanine racemase. No other physiological L-amino acids were effective. Both membrane enzymes and consequent solute transport are markedly reduced in vesicles from glucose-grown cells. Respiratory chain uncouplers abolish the racemase-dehydrogenase-supported transport activity. When amino-oxyacetate at 10-4 M is added to the vesicles, the racemase activity and transport driven by L-alanine and L-serine is specifically and reversibly inhibited. D-Alanine-driven transport is unaffected. Similarly beta-chloro-L-alanine is an irreversible inactivator of the bound racemase but not the D-alanine dehydrogenase. Both the D and L isomers of beta-chloroalanine support oxygen uptake by the vesicles and initially stimulate L-(14C)proline active transport. However, oxidation of the beta-chloro-D-alanine rapidly uncouples active transport from substrate oxidation. This transport inactivation can be protected partially by dithiothreitol, putatively scavenging a reactive product of chloroalanine oxidation. Authentic beta-chloropyruvate produces the same transport uncoupling. When beta-chloro-L-alanine is employed as a substrate, no such transport inactivation is observed. This difference may stem from the possibility that the alanine racemase eliminates HCl from beta-chloro-L-alanine producing pyruvate, not the beta-chloropyruvate that would arise from racemization and then dehydrogenation. We have shown that exogenous pyruvate is oxidized by the vesicles and will also stimulate active transport of amino acids.

Acetates↗
PubMed1976

Alanine and glutamine synthesis and release from skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.

Alanine↗
PubMed1984

Sodium-alanine cotransport in oocytes of Xenopus laevis: correlation of alanine and sodium fluxes with potential and current changes.

The sodium-dependent L-alanine transport across the plasma membrane of oocytes of Xenopus laevis was studied by means of [14C]-L-alanine, 22Na+ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 2:1 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 2:1. The sodium-L-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess of D-alanine over L-alanine caused about 60% inhibition.

Alanine↗
PubMed1988

Effect of alanine concentration independent of changes in insulin and glucagon on alanine and glucose homeostasis in the conscious dog.

The effect of an alanine load per se on hepatic alanine balance and hepatic glucose production is unclear. To examine this question, alanine was infused into six postabsorptive dogs at a rate of 6 mumol/kg-min, while maintaining insulin and glucagon levels using the pancreatic clamp technique. The arterial alanine concentration rose from a basal level of 227 +/- 16 mumol/L to 497 +/- 40 mumol/L during alanine infusion (P less than .01). The net hepatic fractional extraction of alanine remained unchanged, while hepatic alanine uptake increased from 3.0 +/- 0.3 to 6.0 +/- 0.4 mumol/kg-min (P less than .01). Conversion of alanine into glucose increased 87% to 2.7 +/- 0.3 mumol/kg-min during alanine infusion (P less than .01) while gluconeogenic efficiency remained essentially unchanged. Despite the increased gluconeogenic rate, the total rate of glucose production was unchanged. These data suggest that an increase in the alanine load to the liver causes a proportional increase in net hepatic alanine uptake and the gluconeogenic rate, but that in an overnight fasted animal this increase is insufficient to significantly increase glucose production.

Alanine↗
PubMedDate not supplied

Gene transfer efficacies of novel cationic amphiphiles with alanine, beta-alanine, and serine headgroups: a structure-activity investigation.

Herein, we report on the relative in vitro efficacies of nine novel non-glycerol based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and beta-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. The gene transfer efficiencies of lipids 1-9 were evaluated using the reporter gene assays in all the four cell lines and the whole cell histochemical X-gal staining assays in representative CHO cells. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine (4-6) and beta-alanine (7-9) headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts (1-3). Most notably, in CHO, HepG2, and MCF-7 cells, in combination with cholesterol as auxiliary lipid, the transfection efficiencies of the cationic lipids with alanine and beta-alanine headgroups and myristyl and palmityl tails (lipids 4, 5, 7 and 8) were significantly higher (2-3-fold) than that of LipofectAmine-2000, a widely used commercially available liposomal tranfection vectors. Surprisingly, in COS-1 cells, although cationic lipids with beta-alanine headgroups (7-9) were strikingly transfection efficient (3-4-fold more efficacious than LipofectAmine-2000), the gene transfer properties of both their structural isomers (4-6) and their serine headgroup counterparts (1-3) were adversely affected. In summary, the present structure-activity investigation demonstrate that high gene delivery efficacies of cationic amphiphiles containing alanine or beta-alanine headgroups can get seriously compromised by substituting the alanine or beta-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids.

Alanine↗
PubMed1993

Binding of phenol and analogues to alanine complexes of tyrosine phenol-lyase from Citrobacter freundii: implications for the mechanisms of alpha,beta-elimination and alanine racemization.

We have examined the interaction of Citrobacter freundii tyrosine phenol-lyase with both L- and D-alanine. This enzyme catalyzes the racemization of alanine as a side reaction, in addition to the physiological beta-elimination of L-tyrosine to give phenol and ammonium pyruvate. The steady-state kinetic parameters for alanine racemization, kcat and Km, for D-alanine are 0.008 S-1 and 32 mM, respectively, while those for L-alanine are 0.03 S-1 and 11 mM. Incubation of tyrosine phenol-lyase with either L- or D-alanine forms a quinonoid complex that exhibits a strong peak at 500 nm. The presence of K+ increases the intensity of the 500-nm absorption with L-alanine, but decreases the intensity of the peak with D-alanine. Rate constants for the formation of these quinonoid intermediates and the effects of phenol and analogues on the reaction with either L- or D-alanine have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Phenol binds to all the intermediates of tyrosine phenol-lyase with L- and D-alanine, but most strongly to the external aldimine complex, resulting in a decrease in the absorbance at 500 nm at equilibrium. Pyridine N-oxide binds selectively to the quinonoid complex of alanine, and thus causes an increase in the absorbance at 500 nm at equilibrium. 4-Hydroxypyridine causes a decrease in absorbance at 500 nm during the fast phase, but an increase in absorbance at 502 nm in a subsequent slow relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1992

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.

Alanine↗
PubMed1978

D-alanine incorporation into macromolecules and effects of D-alanine deprivation on active transport in Bacillus subtilis.

An auxotroph of Bacillus subtilis 168 unable to synthesize D-alanine loses the ability to support endogenously energized transport when deprived of D-alanine. Revertants of the mutant retain transport activity. The loss of transport is specific for substrates taken up by active transport; substrates taken up by group translocation are transported at normal rates. The loss of transport can be retarded by pretreatment of the cells with inhibitors of protein synthesis. Since the loss of transport could be due to an alteration in a D-alanine-containing polymer, we investigated the incorporation of D-[14C]alanine into macromolecules. The major D-alanine-containing polymers in B. subtilis are peptidoglycan and teichoic acid, with 4 to 6% of the D-[14C]alanine label found in trypsin-soluble material. Whereas the peptidoglycan and teichoic acid undergo turnover, the trypsin-soluble material does not. Treatment of the trypsin-soluble material with Pronase releases free D-alanine. Analysis of acid-hydrolyzed trypsin-soluble material indicated that approximately 75% of the radioactivity is present as D-alanine, with the remainder present as L-alanine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified D-[14C]alanine-labeled membranes indicated the presence of two peaks of radioactivity (molecular weights, 230,000 and 80,000) that could be digested by trypsin. The results suggest that D-alanine may be covalently bound to cellular proteins.

Alanine↗
PubMed1999

beta-alanine and alpha-fluoro-beta-alanine concentrative transport in rat hepatocytes is mediated by GABA transporter GAT-2.

Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of beta-alanine as well as alpha-fluoro-beta-alanine (FbetaAL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na+-dependent transport with high affinity for beta-alanine and GABA with Michaelis constant (Km) of 35.3 and 22.5 microM, respectively. A second Na+-dependent kinetic component with Km >1 mM was also identified. The sigmoidal profile of beta-alanine uptake with respect to Na+ shows the involvement of multiple ions of sodium in the transport process. A Hill coefficient of 2.6 +/- 0.4 indicates that at least two sodium ions are cotransported with beta-alanine. The flux of beta-alanine was also shown to be chlorine dependent. The substitution of this anion with gluconate, even in the presence of Na+, reduced the intracellular concentrative accumulation of beta-alanine to passive diffusion level, indicating that both Na+ and Cl- are essential for the activity of this transporter. The transport of beta-alanine was inhibited by GABA, hypotaurine, beta-aminoisobutyric acid, and FbetaAL in a competitive manner. However, concentrations up to 1 mM of L- and D-alanine, taurine, and alpha-aminoisobutyric acid did not affect beta-alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepatocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 transfected Madin-Darby canine kidney cell extract, confirming the extraneural localization of this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be explained with the formation of beta-alanine and FbetaAL and their effect on the cellular reuptake of GABA.

Animals↗
PubMed1997

Increased alanine uptake and lipid synthesis from alanine in isolated hepatocytes of Wistar-Kyoto fatty rats: an inhibitory effect of biguanides.

To examine the pathophysiological characteristics of non-insulin-dependent diabetes mellitus, alanine metabolism in isolated hepatocytes of male Wistar-Kyoto (WKY) fatty rats (genetically obese and hyperglycemic) and their lean littermates was investigated. The effects of glucagon and the biguanides, metformin and buformin, on alanine metabolism were also studied by measuring alanine uptake and lipid synthesis from alanine. WKY fatty rats showed higher plasma insulin and lipid concentrations than lean rats at 5 as well as at 12 weeks of age. Alanine uptake into hepatocytes was increased in fatty rats only at 12 weeks of age compared with lean rats. Lipid synthesis from alanine in hepatocytes was increased in fatty rats at 5 and 12 weeks of age compared with lean rats. Glucagon increased alanine uptake into hepatocytes but did not affect lipid synthesis from alanine in both fatty and lean rats. Low concentrations (0.1 mM) of biguanides decreased lipid synthesis from alanine only in fatty rats without inhibiting alanine uptake into hepatocytes. These observations suggest that lipid synthesis from alanine in hepatocytes of WKY fatty rats is accelerated prior to the onset of diabetes mellitus, which might be associated with the development of diabetes, and that an inhibitory effect on increased lipid synthesis is one of the pharmacodynamic actions of biguanides.

Aging↗
PubMed1976

D-Alanine dehydrogenase. Its role in the utilisation of alanine isomers as growth substrates by Pseudomonas aeruginosa PA01.

Pseudomonas aeruginosa PA01 was found to utilise both the D- and L-isomers of alpha-alanine and also beta-alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation of D-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation of L-alanine required its conversion to the directly oxidisable D-form by a soluble racemase; (iii) utilisation of beta-alanine, like L-alanine, involves both the racemase and D-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity of which is still speculative; (iv) P. aeruginosa, like Escherichia coli, appears to take up D-alanine and L-alanine by means of two specific permeases.

Alanine↗
PubMed2006

The occurrence of free D-alanine and an alanine racemase activity in Leishmania 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.

Alanine↗
PubMed1976

Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate.

Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.

Alanine↗
PubMed1997

The dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce necrotic- and apoptotic-like death of rat cerebellar granule cells.

The neurotoxic properties of the dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. Beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of beta-N-methylamino-L-alanine were blocked by D(-)-2-amino-5-phosphonopentanoic acid, and those of beta-N-oxalylamino-L-alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K+ (30 mM)- to a low K+ (10 mM)-containing medium. In cerebellar granule cells exposed to beta-N-methylamino-L-alanine or beta-N-oxalylamino-L-alanine (3 mM), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce death over 12-72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.

Amino Acids, Diamino↗
PubMed2006

Allosteric inhibition of Staphylococcus aureus D-alanine:D-alanine ligase revealed by crystallographic studies.

D-alanine:D-alanine ligase (DDl) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. It catalyzes the formation of D-alanine:D-alanine dipeptide, sequentially by using one D-alanine and one ATP as substrates for the first-half reaction, and a second D-alanine substrate to complete the reaction. Some gain of function DDl mutants can use an alternate second substrate, causing resistance to vancomycin, one of the last lines of defense against life-threatening Gram-positive infections. Here, we report the crystal structure of Staphylococcus aureus DDl (StaDDl) and its cocrystal structures with 3-chloro-2,2-dimethyl-N-[4(trifluoromethyl)phenyl]propanamide (inhibitor 1) (Ki=4 microM against StaDDl) and with ADP, one of the reaction products, at resolutions of 2.0, 2.2, and 2.6 A, respectively. The overall structure of StaDDl can be divided into three distinct domains. The inhibitor binds to a hydrophobic pocket at the interface of the first and the third domain. This inhibitor-binding pocket is adjacent to the first D-alanine substrate site but does not overlap with any substrate sites. An allosteric inhibition mechanism of StaDDl by this compound was proposed. The mechanism provides the basis for developing new antibiotics targeting D-alanine:D-alanine ligase. Because this compound only interacts with residues from the first D-alanine site, inhibitors with this binding mode potentially could overcome vancomycin resistance.

Allosteric Regulation↗
PubMed1987

Transport of the alpha-amino-mono-carboxylic acid L-alanine by the beta-alanine carrier of the rabbit ileum.

The proposal that the beta-alanine carrier of the rabbit ileum is a high affinity carrier of the neutral amino acids was examined by means of measurements of influx across the brush border membrane of the intact epithelium using L-alanine as a representative of the neutral amino acids. Confirming the proposal, evidence was provided for mutual competitive inhibition between beta-alanine and L-alanine; and it was also demonstrated that a process contributes to the influx of L-alanine, which is characterized by a maximum rate of transport equal to that of beta-alanine and a Kt, which is equal to the Ki of L-alanine against the influx of beta-alanine. In the concentration range 0.01 to 0.125 mM the influx of L-alanine was found to be linearly related to the concentration indicating a significant unstirred layer influence on present and previous estimates of the Kt values for influx of amino acids across the brush-border membrane of intact intestinal epithelia.

Alanine↗
PubMed2002

Mycobacterium smegmatis D-Alanine Racemase Mutants Are Not Dependent on D-Alanine for Growth.

Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the D-alanine racemase gene (alrA), which is involved in the synthesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM D-alanine and 20 microg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without D-alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of D-alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for D-alanine, suggesting the existence of a new pathway of D-alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable D-alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent D-cycloserine. The MIC of D-cycloserine for the mutant strain was 2.56 microg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of D-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of D-alanine racemase as a target of D-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.

Alanine↗