Latent Tuberculosis Infection (LTBI) and Its Potential Targets: An Investigation into Dormant Phase Pathogens.

One-third of the world's population harbours the latent tuberculosis infection (LTBI) with a lifetime risk of reactivation. Although, the treatment of LTBI relies significantly on the first-line therapy, identification of novel drug targets and therapies are the emerging focus for researchers across the globe. The current review provides an insight into the infection, diagnostic methods and epigrammatic explanations of potential molecular targets of dormant phase bacilli. This study also includes current preclinical and clinical aspects of tubercular infections and new approaches in antitubercular drug discovery.

Lysine ε-aminotransferases: kinetic constants, substrate specificities, and the variation in active site residues.

L-Lysine ε-aminotransferase (lysAT) is an important enzyme in tailoring the terminal amino group of L-lysine or L-ornithine and can be directed to the synthesis of various value-added chemicals such as adipic acid. Three lysATs, lysAT from Saccharopolyspora erythraea NRRL 2338 (lysAT_Sery), lysAT from Nocardia farcinica IFM 10152, and lysAT from Rhodococcus jostii RHA1, were cloned, and their kinetic values and substrate specificities were investigated. In the reaction using 5mM L-lysine and 10mM α-ketoglutarate, lysAT_Sery from S. erythraea NRRL 2338 showed 72% higher specific activity than lysAT from Nocardia farcinica IFM 10152 and 42% higher specific activity than lysAT from R. jostii RHA1. More interesting result was that lysAT Sery, exhibiting the highest activity among three lysATs, did not show any activity to L-ornithine. The alignment of 146 lysAT sequences from RefSeq database was searched by the EC number of lysAT to compare the active site residues among the lysAT sequences. The sequence alignment showed that only two residues, corresponding to Ala129 and Asn328 of lysAT from Mycobacterium tuberculosis H37Rv (lysAT_Mtub), showed variations among the active site residues. All the active site residues except those two residues were completely conserved throughout 145 lysAT sequences. lysAT from S. erythraea NRRL 2338 has A129T and N328S variations (residue numbers are those of the crystal structure of lysAT_Mtub). The structural analysis by the homology model indicate that Thr126 by A129T variation in lysAT_Sery is appeared to interact more tightly with the phosphate group of PLP than alanine (the distance between Thr126 and the phosphate group of PLP was 2.92Å). In addition, Ser328 is located at the substrate recognition site of active site and, therefore, N328S variation may be connected to the substrate specificity of lysAT.

Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance.

Design and Development of Mycobacterium tuberculosis Lysine ɛ-Aminotransferase Inhibitors for Latent Tuberculosis Infection.

Lysine ɛ-aminotransferase (LAT) is a protein involved in lysine catabolism, and it plays a significant role during the persistent/latent phase of Mycobacterium tuberculosis (MTB), as observed by its up-regulation by ~40-fold during this stage. We have used the crystal structure of MTB LAT in external aldimine form in complex with its substrate lysine as a template to design and identify seven lead compounds with IC50 ranging from 18.06 to > 90 µm. We have synthesized 21 compounds based on the identified lead, and compound 21 [2,2'-oxybis(N'-(4-fluorobenzylidene)acetohydrazide)] was found to be the most active with MTB LAT IC50 of 0.81 ± 0.03 µm. Compound 21 also showed a 2.3 log reduction in the nutrient-starved MTB model and was more potent than standard isoniazid and rifampicin at the same dose level of 10 µg/mL.

Discovery of novel lysine ɛ-aminotransferase inhibitors: An intriguing potential target for latent tuberculosis.

Mycobacterium tuberculosis (MTB) has remarkable ability to persist in the human host and causes latent infection in one third of the world population. Currently available tuberculosis (TB) drugs while effective in killing actively growing MTB, is largely ineffective in killing persistent or latent MTB. Lysine-ɛ aminotransferase (LAT) enzyme is reported to be highly up-regulated (41.86 times) in in vitro models of TB designed to mimic the latent stage. Hence inhibition of this MTB LAT seems attractive for developing novel drugs against latent TB. In the present study, crystal structure of the MTB LAT bound to substrate was used as a framework for structure-based design utilizing database compounds to identify novel thiazole derivative as LAT inhibitors. Thirty six compounds were synthesized and evaluated in vitro for their ability to inhibit LAT, in vitro activity against latent MTB, in vivo activity using Mycobacterium marinum infected zebra fish and cytotoxicity as steps toward the derivation of structure-activity relationship (SAR) for lead optimization. Compound 4-methoxy-2-(pyridin-4-yl)thiazole-5-carboxylic acid (24) emerged as the most promising lead with an IC50 of 1.22 ± 0.85 µM against LAT and showed 2.8 log reduction against nutrient starved MTB, with little cytotoxicity at a higher concentration (>50 µM). It also exhibited 1.5 log reduction of M. marinum load in in vivo zebra fish model at 10 mg/kg.

Enzymatic preparation of 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline from (α-N-protected)-L-ornithine using a transaminase or an amine oxidase.

N-Cbz-4,5-dehydro-L-prolineamide or N-Boc-4,5-dehydro-L-prolineamide are alternative key intermediates for the synthesis of saxagliptin, a dipeptidyl peptidase IV (DPP4) inhibitor recently approved for treatment of type 2 diabetes mellitus. An efficient biocatalytic method was developed for conversion of L-ornithine, N-α-benzyloxycarbonyl (Cbz)-L-ornthine, and N-α-tert-butoxycarbonyl (Boc)-L-ornithine to 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline, respectively. Rec. Escherichia coli expressing lysine-ε-aminotransferase and rec Pichia pastoris expressing L-ornithine oxidase were used for these conversions. N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline were chemically converted to key intermediates N-Cbz-4,5-dehydro-L-prolineamide and N-Boc-4,5-dehydro-L-prolineamide, respectively.

Synthesis of 1-piperideine-6-carboxylic acid produced by L-lysine-epsilon-aminotransferase from the Streptomyces clavuligerus gene expressed in Escherichia coli.

The gene (lat) encoding L-lysine epsilon-aminotransferase (LAT) in Streptomyces clavuligerus was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of lat predicted a single open reading frame (ORF) of 1371 bp, encoding a polypeptide of 457 amino acids with calculated molecular mass of 49.89 kDa. S. clavuligerus LAT was grouped into aminotransferase subfamily II of alpha family on the basis of sequence homology. A model system composed of the recombinant LAT in phosphate buffer was set up to study the biosynthesis of 2-acetyltetrahydropyridine. Lysine was found to be transformed to 1-piperideine-6-carboxylic acid. 2-Acetyltetrahydropyridine was characterized from the mixture of 1-piperideine-6-carboxylic acid and methylglyoxal. For the first time, we demonstrated that the L-lysine epsilon-aminotransferase is responsible for the formation of 1-piperideine-6-carboxylic acid, which may react with methylglyoxal to generate the acylated N-heterocyclic odorant 2-acetyltetrahydropyridine.

Identification of the initial steps in D-lysine catabolism in Pseudomonas putida.

Pseudomonas putida uses l-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways delta-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and alpha-aminoadipate are the key metabolites. All the genes and enzymes involved in the d-lysine pathway, except for those involved in the conversion of d-lysine into Delta(1)-piperideine-2-carboxylate, have been identified previously (30). In this study we report that the conversion of d-lysine into Delta(1)-piperideine-2-carboxylate can be mediated by a d-lysine aminotransferase (PP3590) and a d-lysine dehydrogenase (PP3596). From a physiological point of view PP3596 plays a major role in the catabolism of d-lysine since its inactivation leads to a marked reduction in the growth rate with l- or d-lysine as the sole carbon and nitrogen source, whereas inactivation of PP3590 leads only to slowed growth. The gene encoding PP3590, called here amaC, forms an operon with dpkA, the gene encoding the enzyme involved in conversion of Delta(1)-piperideine-2-carboxylate to l-pipecolate in the d-lysine catabolic pathway. The gene encoding PP3596, called here amaD, is the fifth gene in an operon made up of seven open reading frames (ORFs) encoding PP3592 through PP3597. The dpkA amaC operon was transcribed divergently from the operon ORF3592 to ORF3597. Both promoters were mapped by primer extension analysis, which showed that the divergent -35 hexamers of these operon promoters were adjacent to each other. Transcription of both operons was induced in response to l- or d-lysine in the culture medium.

Direct evidence for a glutamate switch necessary for substrate recognition: crystal structures of lysine epsilon-aminotransferase (Rv3290c) from Mycobacterium tuberculosis H37Rv.

Lysine epsilon-aminotransferase (LAT) is a PLP-dependent enzyme that is highly up-regulated in nutrient-starved tuberculosis models. It catalyzes an overall reaction involving the transfer of the epsilon-amino group of L-lysine to alpha-ketoglutarate to yield L-glutamate and alpha-aminoadipate-delta-semialdehyde. We have cloned and characterized the enzyme from Mycobacterium tuberculosisH37Rv. We report here the crystal structures of the enzyme, the first from any source, in the unliganded form, external aldimine with L-lysine, with bound PMP and with its C5 substrate alpha-ketoglutarate. In addition to interaction details in the active site, the structures reveal a Glu243 "switch" through which the enzyme changes substrate specificities. The unique substrate L-lysine is recognized specifically when Glu243 maintains a salt-bridge with Arg422. On the other hand, the binding of the common C5 substrates L-glutamate and alpha-ketoglutarate is enabled when Glu243 switches away and unshields Arg422. The structures reported here, sequence conservation and earlier mutational studies suggest that the "glutamate switch" is an elegant solution devised by a subgroup of fold type I aminotransferases for recognition of structurally diverse substrates in the same binding site and provides for reaction specificity.