Use of NMR metabolomics to analyze the targets of D-cycloserine in mycobacteria: role of D-alanine racemase.

D-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.

Alanine racemase from the green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0-2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 micromol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 degrees C and pH 8.4 and requires pyridoxal 5'-phosphate as a coenzyme.

A truncated Bacillus subtilis dal gene with a 3' ssrA gene tag regulates the growth and virulence of racemase-deficient Listeria monocytogenes.

Listeria monocytogenes (Lm) is a Gram-positive intracellular pathogen that can elicit strong cellular immunity. An attenuated strain (Lmdd) with deletions in two genes (dal and dat) required for d-alanine synthesis and viability has been shown to induce long-lived protective systemic and mucosal immune responses in mice when administered in the presence of the required amino acid. To bypass the necessity for exogenous d-alanine without compromising the safety of the original strain, the defect of Lmdd was complemented with a heterologous Bacillus subtilis dal gene, and the effects of truncating the upstream region of the gene on its transcription efficiency and of modifying its protein product with an ssrA tag at the 3'-terminus were examined. The strains with 551 bp and 80 bp upstream regions showed high levels of transcription and grew without d-alanine. The strains with the shortest upstream regions, 48 bp and 18 bp, showed greatly decreased levels of transcription and failed to grow in the absence of d-alanine. Addition of an ssrA tag to the longer genes resulted in a somewhat altered growth pattern in media and a reduced plaque size on L2 fibroblasts. These bacteria contained low levels of racemase protein and reduced free pools of d-alanine. One of the strains tested further, Lmdd/pA80S, was rapidly cleared from the spleens of infected mice but nevertheless induced a strong immune response that protected mice against challenge by wild-type L. monocytogenes. These bacteria can thus induce immune responses in mice comparable to the original Lmdd strain, but without the need for exogenous d-alanine, and may have use as a live vaccine vector against infectious diseases and cancers.

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli.

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

Directed evolution of bacterial alanine racemases with higher expression level.

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of L-alanine and D-alanine. It can be classified into two groups: biosynthetic enzymes with low catalytic activity and catabolic enzymes with high catalytic activity. It can react with serine to a limited extent. Two biosynthetic alanine racemase genes in Escherichia coli and Salmonella typhimurium were DNA shuffled, and a very diverse chimeric gene library was constructed. An E. coli serine auxotroph was transformed with the shuffled genes, and the recombinant clones were screened on selective media supplemented with 0.5-5 mM D-serine as an L-serine supplier. Selected clones were expected to contain racemases exhibiting higher catalytic activities toward alanine as well as serine. Three independent clones that grew on selective media were isolated. The specific activities of crude extracts prepared from cells expressing the chimeric racemases were increased up to approximately three times more than those expressing the parental enzymes. The best chimera Ser15 racemase was expressed at a level approximately twofold higher than the parental alanine racemases. This high protein expression was demonstrated to be posttranscriptionally achieved.

Roles of Mycobacterium smegmatis D-alanine:D-alanine ligase and D-alanine racemase in the mechanisms of action of and resistance to the peptidoglycan inhibitor D-cycloserine.

D-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of D-alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of L- and D-alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of L- over D-alanine. In all strains, DCS treatment led to a significant accumulation of L-alanine and a concomitant decease of D-alanine, with approximately a 20-fold excess of L-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-alanine branch of mycobacterial peptidoglycan biosynthesis.

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.

Characterization of the alanine racemases from Pseudomonas aeruginosa PAO1.

Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catalyzing the racemization between l- and d-alanine. The requirement for d-alanine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics. In an effort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aeruginosa, an important opportunistic bacterial pathogen of humans and animals. The dadX(PA) and alr(PA) genes have been sequenced, overexpressed, and their activity was demonstrated by complementing d-alanine auxotrophs of Escherichia coli. Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals were obtained.

Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus.

A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.

Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation.

Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase.

A fragment of the Escherichia coli K-12 chromosome complementing the D-amino acid dehydrogenase and catabolic alanine racemase deficiency of a dad operon deletion mutant was cloned in a mini-Mu plasmid. The dadA and dadX genes were localized to a 3.5-kb part of the plasmid insert. The nucleotide sequence of this fragment revealed two open reading frames encoding 432- and 356-amino-acid-long proteins. We show here that they correspond to the dadA and dadX genes. The dadA gene can encode only the smaller of the two subunits of D-amino acid dehydrogenase. A computer search revealed the presence of a flavin adenine dinucleotide-binding motif in the N-terminal domain of the deduced DadA protein sequence. This is in agreement with biochemical data showing that the D-amino acid dehydrogenase contains flavin adenine dinucleotide in its active center. The predicted dadX gene product appeared to be 85% identical to a dadB-encoded catabolic alanine racemase of Salmonella typhimurium. The organization of the dadA and dadX genes confirmed our previous conclusion based on the genetic data (J. Wild, J. Hennig, M. Lobocka, W. Walczak, and T. Klopotowski, Mol. Gen. Genet. 198:315-322, 1985) that these genes form an operon. The main transcription start points of the dad operon were determined by primer extension. They are preceded by a putative sigma 70 promoter sequence and two cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites, one of higher and one of lower affinity to CRP. We propose that the high-affinity site, centered 59.5 bp upstream of the main transcription start point, plays a role in cAMP-CRP-mediated activation of dad operon expression in the absence of glucose.

Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination.

The nucleotide sequence of the alr gene encoding the biosynthetic alanine racemase in Salmonella typhimurium is reported. The sequence was determined by the dideoxy chain termination method of Sanger mostly from recombinants derived from shotgun and specific subcloning of a 2.6-kilobase region containing the alr gene. The final bridging of nonoverlapping contiguous sequences was accomplished with the use of synthetic site-specific primers. The alr gene was found to be 1077 base pairs in length encoding a protein of 359 amino acid residues. Comparison of alr with the dadB gene encoding the catabolic alanine racemase in S. typhimurium revealed almost identical size (1077 vs. 1068 base pairs) and 52% sequence identity. The respective gene products displayed 43% homology, which includes a decapeptide bearing the pyridoxal 5'-phosphate binding site.