Mutational analysis of Mycobacterium tuberculosis lysine ɛ-aminotransferase and inhibitor co-crystal structures, reveals distinct binding modes.

Lysine ɛ-aminotransferase (LAT) converts lysine to α-aminoadipate-δ-semialdehyde in a PLP-mediated reaction. We mutated active-site T330, N328 and E243, and structurally rationalized their properties. T330A and T330S mutants cannot bind PLP and are inactive. N328A although inactive, binds to PLP. E243A retains activity, but binds α-ketoglutarate in a different conformation. We had earlier identified 2-aminomethyl piperidine derivative as a LAT inhibitor. The co-crystal structure reveals that it mimics binding of C5 substrates and exhibits two binding modes. E243, that shields R422 in the apo enzyme, exhibits conformational changes to permit the binding of the inhibitor in one of the binding modes. Structure-based analysis of bound water in the active site suggests optimization strategies for synthesis of improved inhibitors.

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.

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.

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.

Overexpression, purification and crystallization of lysine epsilon-aminotransferase (Rv3290c) from Mycobacterium tuberculosis H37Rv.

Lysine epsilon-aminotransferase (LAT) is a protein involved in lysine catabolism; it belongs to the aminotransferase family of enzymes, which use pyridoxal 5'-phosphate (PLP) as a cofactor. LAT probably plays a significant role during the persistent/latent phase of Mycobacterium tuberculosis, as observed by its up-regulation by approximately 40-fold during this stage. Crystals of recombinant LAT have been grown in 0.1 M trisodium citrate dihydrate solution containing 0.2 M ammonium acetate and 25% PEG 4000 in the pH range 5.4-6.0. Diffraction data extending to 1.98 A were collected at room temperature from a single crystal. Crystals are trigonal in shape and belong to space group P3(1)21, with unit-cell parameters a = 103.26, b = 103.26, c = 98.22 A. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V(M)) of 3.1 A3 Da(-1).

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.

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.

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.

A novel purification procedure of L-lysine 6-aminotransferase from Flavobacterium lutescence.

A new method for the purification of L-lysine 6-aminotransferase (L-lysine: 2-oxoglutarate 6-aminotransferase, EC 2.6.1.36) was devised, in which affinity chromatography with L-lysylacetamidododecyl-Sepharose 6B, the most effective affinity adsorbent, was substituted for the heat treatment. The yield of the enzyme with the present procedure was approx. twice as high as that with the previous procedure (Soda, K. and Misono, H. (1968) Biochemistry 7, 4110-4119). The enzyme purified by this method was activated 2-fold by heat treatment (65 degrees C for 5 min). The enzyme has absorption maxima at 340 and 415 nm, derived from the bound pyridoxal 5'-phosphate, which are identical with those of the enzyme obtained with the procedure including heat treatment. These results rule out the possibility that the formation of the 340-nm pyridoxal 5'-phosphate of the enzyme is an artifact of heat treatment.

[Effect of lat disruption on clavulanic acid production].

A 1.8kb fragment of lat was obtained from Streptomyces clavuligerus 27064, and replacement plasmid of pXAL1 and pXAL2 were constructed. PXAL1 and pXAL2 were used to disrupt the lat gene by bi-parental conjugation from E. coli to Streptomyces clavuligerus. A Am(r)Thio(S) transformant, named as XAL863, was obtained. The genome of Streptomyces clavuligerus 27064 and XAL863 was analyzed by southern blot technique, and the activity of lysine epsilon-aminotransferase in the two strains was also tested. Both results proved that the lat was disrupted in the XAL863. Streptomyces clavuligerus and XAL863 were cultured in the shaken flask respectively, and the production of clavulanic acid was analyzed by HPLC with the different incubation time interval, and the yield was approximately 1.8 times higher in the XAL863 at their highest production point.

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.

Transcriptional mapping of the genes encoding the early enzymes of the cephamycin biosynthetic pathway of Streptomyces clavuligerus.

Isopenicillin-N synthase (IPNS) of Streptomyces clavuligerus is encoded by the pcbC gene which is found within the cephamycin biosynthetic gene cluster. pcbC is located directly downstream from lat and pcbAB, which encode the enzymes, lysine epsilon-amino transferase and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, respectively. These enzymes act prior to IPNS in the biosynthetic pathway, and the three genes are transcribed in the same direction. Previous pcbC transcriptional studies involving recombinant promoter probe plasmids, Northern analysis and 5' primer extension indicated the presence of a monocistronic 1.2-kb transcript that initiated within pcbAB, 92-bp upstream from the pcbC start codon. S1 nuclease mapping studies have now shown, not only the transcript initiating 92 bp upstream from pcbC, but also a transcript initiating further upstream, possibly including the entire pcbAB gene. Promoter probe analysis and S1 nuclease mapping failed to detect promoter activity or a transcription start point (tsp) directly upstream from pcbAB, suggesting that pcbAB transcripts initiated within or upstream from lat. Northern analysis, to search for a pcbAB transcript, showed no distinct transcript and indicated severely degraded mRNA. Similar results were obtained when Northern analysis was used to search for lat transcripts. Promoter probe analysis to locate the lat promoter indicated that a sequence promoting transcription was present in a 330-bp DNA fragment that extended from 227-bp upstream from the lat structural gene to 103 bp inside the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the half and overall reactions catalyzed by L-lysine:2-oxoglutarate 6-aminotransferase.

Significant differences were found in the reaction rate, and the substrate and reaction specificities between the half reactions and the overall reactions catalyzed by L-lysine: 2-oxoglutarate 6-aminotransferase. The half reactions between an amino donor and the enzyme-bound pyridoxal 5'-phosphate, and also between an amino acceptor and the bound pyridoxamine 5'-phosphate followed first order reaction kinetics. The extrapolated first order rate constants and dissociation constants of the substrates were determined for the half reactions: lysine, 0.87 min-1 and 5.5 mM; glutamate, 1.1 min-1 and 10.5 mM; alanine, 0.66 min-1 and 6.6 mM; 6-aminohexanoate, 0.43 min-1 and 13.3 mM; and 2-oxoglutarate, 0.33 min-1 and 2.5 mM. As compared with the values reported for the overall reactions [Soda, K., Misono, H., & Yamamoto, T. (1968) Biochemistry 7, 4102-4109], the reactivity of the inherent substrates was lower by over 4 orders in the half reaction than that in the overall reaction, and the reactivity of alanine with the bound pyridoxal 5'-phosphate was reduced to 10% of that in the overall reaction. The substrate specificity in the half reaction was much lower than that in the overall reaction, which was re-examined in a reaction system containing the same concentration of the enzyme as that for the half reactions. Lysine 6-aminotransferase catalyzes the transfer of only the terminal amino group of lysine to 2-oxoglutarate in the overall reaction. However, in the half reaction, the 2-amino group as well as the terminal one was transferred to the bound pyridoxal 5'-phosphate. The ratio of reactivity of the 2-amino group to that of the 6-amino group was considerably influenced by the pH of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

L-Lysine: 2-oxoglutarate 6-aminotransferase. Subunit structure composed of non-identical polypeptides and pyridoxal 5'-phosphate-binding subunit.

L-Lysine:2-oxoglutarate 6-aminotransferase from Flavobacterium lutescence (= Achromobacter liquidum) has been shown to be composed of one each of four non-identical subunits, A, B1, B2, and C. The subunits were isolated by gel filtration, and DEAE-cellulose chromatography in the presence of 8 M urea. Their molecular weights were determined by ultracentrifugation, gel electrophoresis and gel filtration: subunit A 24,000; B1 28,000; B2 28,000; C 45,000. These subunits were all different in amino acid composition. Of the two molecules of bound pyridoxal 5'-phosphate, the one which absorbs at 415 nm is bound to subunit B2 and participates in the catalytic action of the enzyme.

The effect of carboxylates and halides on L-lysine 6-aminotransferase-catalyzed reactions.

L-Lysine:2-oxoglutarate 6-aminotransferase catalyzes very slow transamination between L-alanine and 2-oxoglutarate. A high concentration of anions such as formate, acetate and halides greatly accelerated this transamination without affecting the affinity of the enzyme for L-alanine. In contrast, the anions strongly inhibited the normal L-lysine 6-transamination in a competitive manner with L-lysine and in a non-competitive manner with 2-oxoglutarate. This result suggests that the enzyme has an anion binding site which normally binds the carboxyl group of L-lysine. The binding of halides or carboxylates to this site probably induces a conformational change of the enzyme, and results in the inhibition of L-lysine 6-transamination, and in the stimulation of L-alanine transamination. Treatment of the enzyme with an arginine-specific dicarbonyl reagent, phenylglyoxal, led to a loss of the enzyme activity for L-lysine. The activity for L-alanine was not affected, but the stimulating effect of anions on L-alanine transamination was impaired. Thus, it is suggested that an arginine residue(s) plays an important role in the anion binding site.

Characterization of L-lysine 6-aminotransferase and its structural gene from Flavobacterium lutescens IFO3084.

L-Lysine 6-aminotransferase (LAT) is an enzyme involved in L-lysine catabolism in a wide range of living organisms. LAT from Flavobacterium lutescens IFO3084 was purified, and its structural gene (lat) was cloned, sequenced and expressed in Escherichia coli. Native PAGE analysis of purified LAT gave a single band corresponding to a molecular weight of about 110,000. lat encoded a protein of 493 amino acids with a deduced molecular weight of 53,200, which is very close to that of purified LAT determined on SDS-PAGE. Expression of lat in E. coli revealed that lat encodes a single subunit protein leading to LAT activity. These data suggested that LAT from F. lutescens IFO3084, like most other aminotransferases, is derived from a single ORF and is active as a homodimer.

Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug.

Biocatalytic processes were used to prepare chiral intermediates required for the synthesis of Omapatrilat 1 by three different routes. The synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 3 to L-6-hydroxynorleucine 2 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of the ketoacid 3, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 4] with D-amino acid oxidase from porcine kidney or Trigonopsis variabilis followed by reductive amination to convert the mixture completely to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess (e.e.). The enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 5) was demonstrated using phenylalanine dehydrogenase (PDH) from T. intermedius. Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris. Using PDH from E. coli or P. pastoris, the enzymatic process was scale-up to prepare kg quantity of allysine ethylene acetal 5. The reaction yields of >94% and e.e. of >98% were obtained for allysine ethylene acetal 5. An enzymatic process was developed for the synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4 [[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid [BMS-199541-01]. The enzymatic oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[[(phenyl-methoxy)carbonyl] L-homocysteinyl] L-lysine)-1,1-disulphide [BMS-201391-01] to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase (LAT) from Sphingomonas paucimobilis SC 16113 was demonstrated. This enzyme was overexpressed in E. coli and a process was developed using the recombinant enzyme.

Interdependence of gene expression for early steps of cephalosporin synthesis in Streptomyces clavuligerus.

The early steps of cephamycin synthesis by S. clavuligerus are catalyzed sequentially by lysine epsilon-aminotransferase (LAT), delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (cyclase, IPNS). The genes (lat, pcbAB, and pcbC, respectively) are closely linked in the same order as the enzymes act in the biosynthetic pathway and are transcribed in the same direction. Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity. All four mutants are complemented in cephamycin formation by transformation with pNBR1, a plasmid containing a 7.2-kb genomic region of S. clavuligerus in vector pIJ702. The cloned DNA was found to possess no part of the cyclase gene, but instead it contained lat and the 5' upstream part of pcbAB. Doran et al. reported that the 31-bp region between pcbAB and pcbC contains no recognizable promoter or transcription termination sequences. We found that there are 153 bp between the lat ORF and the pcbAB start codon. A potential transcriptional terminator begins 4 to 6 bp downstream of the lat ORF. In the 111-bp segment between the end of the "terminator" and the pcbAB start codon, there are no Streptomyces-like or Escherichia coli-like promoter consensus sequences. However, upstream of the "terminator," that is, in the downstream portion of the lat ORF, are two regions resembling a Streptomyces consensus promoter. Promoter activity in gene fusion constructions was demonstrated in this region. A third potential promoter is upstream of the lat ORF, but only the--10 part is on the cloned DNA. The mechanism by which the cloned DNA (containing lat, the 5' part of pcbAB, and the intervening sequence) influences the expression of the downstream genes encoding ACVS and IPNS, even in strains that possess LAT activity, is an intriguing target of future investigation.

Metabolic engineering of cephalosporin biosynthesis in Streptomyces clavuligerus.

The biosynthesis of beta-lactams is one of the most thoroughly studied antibiotic pathways. The availability of the characteristics and the time profiles of activities of enzymes involved in the biosynthesis allows one to critically evaluate the potential rate-limiting steps in its production. Our approach to understanding the control of beta-lactam biosynthesis has been pursued using a two-stage strategy: (1) to predict the rate-limiting steps using a kinetic model and (2) to relax the rate-limiting steps by engineering the biosynthetic pathway or by altering the kinetic parameters of the predicted key rate-limiting enzyme. Kinetic analysis of the pathway dynamics of cephamycin C production in Streptomyces clavuligerus was performed using data obtained from wild type. Sensitivity analysis revealed that the availability of precursor alpha-aminoadipic acid and activity of ACV synthetase were the potential rate-limiting steps. Relaxation of the precursor limitation was accomplished by integration of an additional copy of the gene encoding lysine-epsilon-aminotransferase (lat) into the chromosome. The recombinant strain showed an increased level of cephamycin C production as expected. The intracellular levels of different intermediates in the pathway in batch cultures were analyzed.

Induction of L-lysine epsilon-aminotransferase by L-lysine in Streptomyces clavuligerus, producer of cephalosporins.

L-Lysine epsilon-aminotransferase (LAT) catalyzes the first reaction in the two-step conversion of L-lysine (Lys) to 1-alpha-aminoadipic acid (Aaa), a direct precursor of cephalosporins (including cephamycin C) in Streptomyces clavuligerus. Previous work showed that addition of Lys to chemically defined medium improved antibiotic production. We show that in S. clavuligerus cultures supplemented with high concentrations of Lys, Lys enhances antibiotic production by a dual effect, i.e. as a substrate of LAT thus providing Aaa and also as an inducer of LAT yielding even more Aaa. On the other hand, LAT is not induced by Aaa.