Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.     

Characterization of Mycobacterium smegmatis expressing the Mycobacterium tuberculosis fatty acid synthase I (fas1) gene

Unlike most other bacteria, mycobacteria make fatty acids with the multidomain enzyme eukaryote-like fatty acid synthase I (FASI). Previous studies have demonstrated that the tuberculosis drug pyrazinamide and 5-chloro-pyrazinamide target FASI activity. Biochemical studies have revealed that in addition to C(16:0), Mycobacterium tuberculosis FASI synthesizes C(26:0) fatty acid, while the Mycobacterium smegmatis enzyme makes C(24:0) fatty acid. In order to express M. tuberculosis FASI in a rapidly growing Mycobacterium and to characterize the M. tuberculosis FASI in vivo, we constructed an M. smegmatis Deltafas1 strain which contained the M. tuberculosis fas1 homologue. The M. smegmatis Deltafas1 (attB::M. tuberculosis fas1) strain grew more slowly than the parental M. smegmatis strain and was more susceptible to 5-chloro-pyrazinamide. Surprisingly, while the M. smegmatis Deltafas1 (attB::M. tuberculosis fas1) strain produced C(26:0), it predominantly produced C(24:0). These results suggest that the fatty acid elongation that produces C(24:0) or C(26:0) in vivo is due to a complex interaction among FASI, FabH, and FASII and possibly other systems and is not solely due to FASI elongation, as previously suggested by in vitro studies.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase

Tuberculosis, which is caused by Mycobacterium tuberculosis, remains to be a global health problem. The thick and complex cell envelope has been implicated in many aspects of the pathogenicity of M. tuberculosis. M. tuberculosis UDP-glucose pyrophosphorylase (UGP, coded by galU, Rv0993) is involved in cell envelope precursor synthesis. UGP catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose 1-phosphate (Glc-l-P). Bacterial UGPs are completely unrelated to their eukaryotic counterparts. This enzyme is recognized as a virulence factor in several bacterial species and is conserved among mycobacterial species, which makes it a good target for mycobacterial pathogenicity research. The recombinant M. tuberculosis UGP (rMtUGP) was purified in Escherichia coli and found to be stable and catalytically active. The effects of pH, temperature and Mg2+ on enzyme activity were characterized. In addition, subcellular localization studies revealed that most of M. tuberculosis UGP protein was located in the cell wall. The purification and characterization of M. tuberculosis UGP may help to decipher the pathogenicity of M. tuberculosis.     

Recombinant BCG as a vaccine vehicle to protect against tuberculosis

Mycobacterium bovis Bacille Calmette Guérin (BCG) was first administered to humans in 1921 and has subsequently been delivered to an estimated 3 billion individuals, with a low incidence of serious complications. The vaccine is immunogenic and is stable and cheap to produce. Additionally, the vaccine can be engineered to express foreign molecules in a functional form, and this has driven the development of BCG as a recombinant vector to protect against infectious diseases and malignancies such as cancer. However, it is now clear that the existing BCG vaccine has proved insufficient to control the spread of tuberculosis, and a major focus of tuberculosis vaccine development programs is the construction and testing of modified forms of BCG. This review summarizes the strategies employed to develop recombinant forms of BCG and describes the potential of these vaccines to stimulate protective immunity and protect against Mycobacterium tuberculosis infection.     

Structure of Streptomyces maltosyltransferase GlgE, a homologue of a genetically validated anti-tuberculosis target

GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (β/α)(8) fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.     

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is a link between the Escherichia coli and mammalian enzymes

D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize alpha-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I(0.5) = 30 microm. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.     

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.     

Inositol-1-phosphate synthetase mRNA as a new target for antisense inhibition of Mycobacterium tuberculosis

The need for novel antimicrobial agents to combat the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis is a worldwide urgency. This study has investigated the effects on phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of inositol-1-phosphate synthase, the key enzyme in the first step in inositol synthesis. Inositol is utilized by M. tuberculosis in the production of its major thiol, which is an antioxidant that helps M. tuberculosis to get rid of reactive oxygen species and electrophilic toxins. Real-time RT-PCR analysis revealed that mRNA expression of inositol-1-phosphate (I-1-P) synthase was significantly reduced upon addition of 20 microM PS-ODNs. Treatment with antisense PS-ODNs also reduced the level of mycothiol and the proliferation of M. tuberculosis and enhanced susceptibility to antibiotics. The experiments indicated that the antisense PS-ODNs could enter the cytoplasm of M. tuberculosis and inhibit the expression of I-1-P synthase. This study demonstrates that the M. tuberculosis I-1-P synthase is a target for the development of novel antibiotics and PS-ODN to I-1-P synthase is a promising antimycobaterial candidate.     

Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site

Ribose-5-phosphate isomerases (EC 5.3.1.6) inter-convert ribose-5-phosphate and ribulose-5-phosphate. This reaction allows the synthesis of ribose from other sugars, as well a means for salvage of carbohydrates after nucleotide breakdown. Two unrelated types of enzyme are known to catalyze the isomerization. The most common one, RpiA, is present in almost all organisms. The second type, RpiB, is found in many bacterial species.Here, we demonstrate that the RpiB from Mycobacterium tuberculosis (Rv2465c) has catalytic properties very similar to those previously reported for the Escherichia coli RpiB enzyme. Further, we report the structure of the mycobacterial enzyme, solved by molecular replacement and refined to 1.88A resolution. Comparison with the E.coli structure shows that there are important differences in the two active sites, including a change in the position and nature of the catalytic base. Sequence comparisons reveal that the M.tuberculosis and E.coli RpiB enzymes are in fact representative of two distinct sub-families. The mycobacterial enzyme represents a type found only in actinobacteria, while the enzyme from E.coli is typical of that seen in many other bacterial proteomes. Both RpiBs are very different from RpiA in structure as well as in the construction of the active site. Docking studies allow additional insights into the reactions of all three enzymes, and show that many features of the mechanism are preserved despite the different catalytic components.     

Expression of the naphthoate synthase gene in Mycobacterium tuberculosis in a self-generated oxygen depleted liquid culture system

Mycobacterium tuberculosis has been classified for decades as a strict aerobic species. Whole genome sequencing of the type culture strain H37Rv has revealed the presence of a full set of genes allowing for anaerobic metabolism. Naphthoate synthase (menB) is a key enzyme required for the synthesis of menaquinone, which plays a crucial role in anaerobic electron transport, ultimately resulting in the formation of energy generating intermediates. Interrupting the synthesis of this enzyme will interfere with the production of menaquinone and therefore this enzyme is a potential drug target. This study serves to investigate the role of naphtoate synthase in the survival of M. tuberculosis H37Rv when incubated under oxygen limiting conditions of unagitated liquid culture over 15 weeks. M.?tuberculosis H37Rv was grown in Middlebrook 7H9 media. The tubes were kept undisturbed at 37?°C for up to 15 weeks. At selected time points, aliquots of cells were removed and frozen. RNA was simultaneously extracted from all aliquots. The RNA was converted to cDNA for Real-Time PCR on the ABI 7000 SDS. Gene expression was normalized against 16S RNA quantities at each time point. A systematic increase in the expression of the menB gene product was observed over the incubation period with a 4.3-fold increase seen at week 6 (P?相似文献   

Detection and purification of a catalase-peroxidase from Mycobacterium sp. Pyr-1

A catalase-peroxidase from Mycobacterium sp. Pyr-1, a strain capable of growth on pyrene, was purified to homogeneity by anion exchange and hydroxyapatite column chromatography. The enzyme, like the M. tuberculosis T-catalase, reduced nitroblue tetrazolium in the presence of isoniazid (INH) and H2O2. It also oxidized 3,3',5,5'-tetramethylbenzidine and other substrates of the catalase-peroxidase of M. tuberculosis in the presence of either tert-butyl hydroperoxide or H2O2. It had a UV/ visible absorption spectrum (Soret peak at 406 nm) similar to that of the catalase-peroxidase of M. tuberculosis (Soret peak at 408 nm) and identical to that of the catalase-peroxidase of M. smegmatis. After electrophoresis on non-denaturing gels the enzyme showed one single protein band with both catalase and peroxidase activity, which were lost after electrophoresis on SDS-PAGE. The enzyme was inhibited by sodium azide, glutathione, 2-mercaptoethanol, and isoniazid, but not by isonicotinic acid. The optimum enzyme activity was obtained at pH 4.5 and at 25 degrees C.     

The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages

Mycobacterium tuberculosis generally reside in phagosomes within human macrophages that resist maturation and acidification, but exhibit significant heterogeneity. In this study we have constructed an IPTG-inducible GFP expression system in M. tuberculosis to assess the relationship between the metabolic status of M. tuberculosis and the degree of phagosomal maturation. Using these recombinant bacteria, we have found that, in human macrophages, M. tuberculosis that respond to IPTG with expression of GFP fluorescence, and hence are metabolically active, reside in non-acidified phagosomes that have not fused with Texas red dextran pre-labelled lysosomes. In contrast, M. tuberculosis that fail to express GFP in response to IPTG, and hence are metabolically inactive, reside within acidified phagosomes that have fused with Texas red dextran labelled lysosomes. These studies demonstrate that metabolic activity of M. tuberculosis correlates strongly with phagosomal maturation and that the inducible GFP expression system is useful for assessing metabolic activity of intracellular M. tuberculosis.     

Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis.

The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to other bacterial catalase-peroxidases. Expression of the katG gene in M. tuberculosis, M. smegmatis, and Escherichia coli was demonstrated by Western blotting (immunoblotting). Homologous genes were detected in other mycobacteria, even those which are naturally insensitive to isoniazid.     

Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.     

Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase

UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU protein involved in the formation of UDP-GlcNAc from other bacteria. GlmU is a bifunctional enzyme that catalyzes two sequential steps in UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl transferase catalyzes the formation of N-acetylglucosamine-1-phosphate, and N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of UDP-GlcNAc. Since inhibition of peptidoglycan synthesis often results in cell lysis, M. tuberculosis GlmU is a potential anti-tuberculosis (TB) drug target. In this study we cloned M. tuberculosis Rv1018c (glmU gene) and expressed soluble GlmU protein in E. coli BL21(DE3). Enzymatic assays showed that M. tuberculosis GlmU protein exhibits both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridylyltransferase activities. We also investigated the effect on Mycobacterium smegmatis when the activity of GlmU is fully removed or reduced via a genetic approach. The results showed that activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU enzyme. As the amount of functional GlmU enzyme was gradually reduced in a temperature shift experiment, the M. smegmatis cells became non-viable and their morphology changed from a normal rod shape to stubby-rounded morphology and in some cases they lysed. Finally a microtiter plate based assay for GlmU activity with an OD340 read out was developed. These studies therefore support the further development of M. tuberculosis GlmU enzyme as a target for new anti-tuberculosis drugs.     

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics.     

A new subfamily of short bacterial adenylate kinases with the Mycobacterium tuberculosis enzyme as a model: A predictive and experimental study.

The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of "short" bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8 degrees C). Based on various spectroscopic data, on the known three-dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238-248.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

Purification and characterization of a functionally active Mycobacterium tuberculosis prephenate dehydrogenase

Tuberculosis (TB) remains to be a global health problem. New drugs are badly needed to drastically reduce treatment time and overcome some of the challenges with tuberculosis treatment, such as multi-drug resistant (MDR) strain infected patients or tuberculosis/HIV co-infected patients. The essentiality of mycobacterial aromatic amino acid biosynthesis pathways and their absence from human host indicate that the member enzymes of these pathways promising drug targets for therapeutic agents against pathogen mycobacteria. Prephenate dehydrogenase (PDH) is a key regulatory enzyme in tyrosine biosynthesis, catalyzing the NAD(+)-dependent conversion of prephenate to p-hydroxyphenylpyruvate, making it a potential drug target for antibiotics discovery. The recombinant PDH with an N-terminal His-tag (His-rMtPDH) was first purified in Escherichia coli, and using enterokinase rMtPDH was obtained by cleaving the N-terminal fusion partner. The effect of pH, temperature and the cation-Na(+) on purified enzyme activity was characterized. The N-terminal fusion partner was found to have little effect on the biochemical properties of PDH. We also provide in vitro evidence that Mycobacterium tuberculosis PDH does not possess any chorismate mutase (CM) activity, which suggests that, unlike many other enteric bacteria (where PDH exists as a fusion protein with CM), M. tuberculosis PDH is a monofunctional protein.