Related Articles

Synopsis Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in Mycobacterium spp. Here we report the crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP at 2.6 Å and 3.0 Å resolution, respectively.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active enZymes data base; see www.cazy.org). Here we report the purification, crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP. GpgS crystals belong to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c= 127.64 Å, and diffract to 2.6 Å resolution. GpgS-UDP complex crystals belong to space group I4 with unit-cell parameters a= 98.32, b= 98.32, c= 127.96 Å, and diffract to 3.0 Å resolution.

Mycobacteria synthesize intracellular methylglucose lipopolysaccharides (MGLP) proposed to regulate fatty acid synthesis. Although their structures have been elucidated, the identity of most biosynthetic genes remains unknown. The first step in MGLP biosynthesis is catalyzed by a glucosyl-3-phosphoglycerate synthase (GpgS, Rv1208 in Mycobacterium tuberculosis H37Rv). However, a typical glucosyl-3-phosphoglycerate phosphatase (GpgP, EC3.1.3.70) for dephosphorylation of glucosyl-3-phosphoglycerate to glucosylglycerate, was absent from mycobacterial genomes. We purified the native GpgP from Mycobacterium vanbaalenii and identified the corresponding gene deduced from amino acid sequences by mass spectrometry. The M. tuberculosis ortholog (Rv2419c), annotated as a putative phosphoglycerate mutase (PGM, EC5.4.2.1), was expressed and functionally characterized as a new GpgP. Regardless of the high specificity for glucosyl-3-phosphoglycerate, the mycobacterial GpgP is not a sequence homolog of known isofunctional GpgPs. The assignment of a new function in M. tuberculosis genome expands our understanding of this organism's genetic repertoire and of the early events in MGLP biosynthesis.

A single-step pathway for the synthesis of the compatible solute glucosylglycerate (GG) is proposed based on the activity of a recombinant glucosylglycerate synthase (Ggs) from Persephonella marina. The corresponding gene encoded a putative glycosyltransferase that was part of an operon-like structure which also contained the genes for glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP), the enzymes that lead to the synthesis of GG through the formation of glucosyl-3-phosphoglycerate. The putative glucosyltransferase gene was expressed in Escherichia coli, and the recombinant product catalyzed the synthesis of GG in one step from ADP-glucose and d-glycerate, with Km values at 70°C of 1.5 and 2.2 mM, respectively. This glucosylglycerate synthase (Ggs) was also able to use GDP- and UDP-glucose as donors to form GG, but the efficiencies were lower. Maximal activity was observed at temperatures between 80 and 85°C, and Mg2+ or Ca2+ was required for catalysis. Ggs activity was maximal and remained nearly constant at pH values between 5.5 and pH 8.0, and the half-lives for inactivation were 74 h at 85°C and 8 min at 100°C. This is the first report of an enzyme catalyzing the synthesis of GG in one step and of the existence of two pathways for GG synthesis in the same organism.

The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adapted bacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, which were separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgS protein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and from d-3-phosphoglycerate, with maximal activity at 90°C. The recombinant GpgP protein, like the M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and “Dehalococcoides ethenogenes.” GpgS had maximal activity at 50°C. The maximal activity of GpgP was at 50°C with GPG as the substrate and at 60°C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme's preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.

Background

Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides - the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) - the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.

Methodology/Principal Findings

We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.

Conclusions/Significance

This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.

N-Acetylglucosamine 1-phosphate uridyltransferase (GlmU) from M. tuberculosis H37Rv has been crystallized and preliminary X-ray crystallographic analysis has been performed. GlmU is a bi-domained bifunctional enzyme that is involved in the biosynthesis of UDP-N-acetylglucosamine, a precursor in peptidoglycan biosynthesis in M. tuberculosis.

The gene product of open reading frame Rv1018c from Mycobacterium tuberculosis is annotated as encoding a probable N-acetylglucosamine 1-­phosphate uridylyltransferase (MtbGlmU), an enzyme that catalyzes the biosynthesis of UDP-N-acetylglucosamine, a precursor common to lipopolysaccharide and peptidoglycan biosynthesis. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. Native diffraction data were collected from crystals belonging to space group R32 and processed to a resolution of 2.2 Å.

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the β-d-glucosyl-(1→6)-d-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose β-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose β-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose → MGA + UDP, and (ii) MGA + UDP-glucose → DGA + UDP. Both enzymes required the addition of K+ ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl2, in contrast to MGA synthase, which did not require Mg2+. It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg2+ requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg2+ requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg2+ increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.

M. tuberculosis tetrahydrodipicolinate-N-succinyltransferase, the enzyme that catalyses the fifth reaction step of the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized.

Tetrahydrodipicolinate-N-succinyltransferase from Mycobacterium tuberculosis (DapD, Rv1201c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in the cubic space group I23 or I213. Preliminary diffraction data analysis indicates the presence of five molecules per asymmetric unit. Furthermore, the data exhibit icosahedral point-group symmetry. One possible explanation for this is that the enzyme assembles into a 60-mer exhibiting 235 point-group symmetry and crystallizes as such in space group I23. In this case, the combination of crystallographic and noncrystallographic symmetry elements results in an arrangement of the icosahedrons in the cubic crystal with one pentamer in the asymmetric unit. Another explanation is that the packing of the molecules itself mimics icosahedral symmetry. In this case both space groups I23 and I213 would be possible.

The small regulatory subunit of acetohydroxylate synthase (IlvH) from M. tuberculosis has been crystallized and preliminary crystallographic analysis has been performed.

Branched amino-acid biosynthesis is important to bacterial pathogens such as Mycobacterium tuberculosis (Mtb), a microorganism that presently causes more deaths in humans than any other prokaryotic pathogen (http://www.who.int/tb). In this study, the molecular cloning, expression, purification, crystallization and preliminary crystallographic analysis of recombinant IlvH, the small regulatory subunit of acetohydroxylic acid synthase (AHAS) in Mtb, are reported. AHAS carries out the first common reaction in the biosynthesis of valine, leucine and isoleucine. AHAS is an essential enzyme in Mtb and its inactivation leads to a lethal phenotype [Sassetti et al. (2001 ▶), Proc. Natl Acad. Sci. USA, 98, 12712–12717]. Thus, inhibitors of AHAS could potentially be developed into novel anti-Mtb therapies.

The acyl-CoA carboxylase β subunit (ACCD6) of M. tuberculosis has been crystallized and preliminary X-ray crystallographic analysis has been performed.

Mycobacterium tuberculosis (Mtb) acyl-CoA carboxylase is involved in the biosynthesis of mycolic acids, which are a key component of the bacillus cell wall. The Mtb genome encodes six acyl-CoA carboxylase β subunits (ACCD1–6), three of which (ACCD4–6) are essential for survival of the pathogen on minimal medium. Mtb ACCD6 has been expressed, purified and crystallized. The two forms of Mtb ACCD6 crystals belonged to space groups P41212 and P212121 and diffracted to 2.9 and 2.5 Å resolution, respectively, at a synchrotron-radiation source.

Mannosyl-3-phosphoglycerate synthase (MpgS) is a key enzyme in the biosynthesis of MG. Here, the purification, crystallization and preliminary crystallographic characterization of apo MpgS from Thermus thermophilus HB27 are reported.

Mannosylglycerate (MG) is a compatible solute that is widespread in marine organisms that are adapted to hot environments, with its intracellular pool generally increasing in response to osmotic stress. These observations suggest that MG plays a relevant role in osmoadaptation and thermoadaptation. The pathways for the synthesis of MG have been characterized in a number of thermophilic and hyperthermophilic organisms. Mannosyl-3-phosphoglycerate synthase (MpgS) is a key enzyme in the biosynthesis of MG. Here, the purification, crystallization and preliminary crystallographic characterization of apo MpgS from Thermus thermophilus HB27 are reported. The addition of Zn2+ to the crystallization buffer was essential in order to obtain crystals. The crystals belonged to one of the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = b = 113, c = 197 Å. Diffraction data were obtained to a resolution of 2.97 Å.

Lewis x (Lex) and sialyl Lewis x (SLex)-containing glycans play important roles in numerous physiological and pathological processes. The key enzyme for the final step formation of these Lewis antigens is α1-3-fucosyltransferase. Here we report molecular cloning and functional expression of a novel Helicobacter hepaticus α1-3-fucosyltransferase (HhFT1) which shows activity towards both non-sialylated and sialylated Type II oligosaccharide acceptor substrates. It is a promising catalyst for enzymatic and chemoenzymatic synthesis of Lex, sialyl Lex and their derivatives. Unlike all other α1-3/4-fucosyltransferases characterized so far which belong to Carbohydrate Active Enzyme (CAZy, http://www.cazy.org/) glycosyltransferase family GT10, the HhFT1 shares protein sequence homology with α1-2-fucosyltransferases and belongs to CAZy glycosyltransferase family GT11. The HhFT1 is thus the first α1-3-fucosyltransferase identified in the GT11 family.

The regulatory domain of M. tuberculosis aspartokinase, the enzyme which catalyses the first reaction step in the biosynthesis of the amino acids lysine, methionine and threonine, has been cloned, expressed, purified and crystallized. Preliminary X-ray diffraction analysis of several crystals revealed the presence of five distinct crystal forms.

The regulatory domain of Mycobacterium tuberculosis aspartokinase (Mtb-AK, Mtb-Ask, Rv3709c) has been cloned, heterologously expressed in Escherichia coli and purified using standard chromatographic techniques. Screening for initial crystallization conditions using the regulatory domain (AK-β) in the presence of the potential feedback inhibitor threonine identified four conditions which yielded crystals suitable for X-ray diffraction analysis. From these four conditions five different crystal forms of Mtb-AK-β resulted, three of which belonged to the orthorhombic system, one to the tetragonal system and one to the monoclinic system. The highest resolution (1.6 Å) was observed for a crystal form belonging to space group P212121, with unit-cell parameters a = 53.70, b = 63.43, c = 108.85 Å and two molecules per asymmetric unit.

The 'global public good' (GPG) concept has gained increasing attention, in health as well as development circles. However, it has suffered in finding currency as a general tool for global resource mobilisation, and is at risk of being attached to almost anything promoting development. This overstretches and devalues the validity and usefulness of the concept. This paper first defines GPGs and describes the policy challenge that they pose. Second, it identifies two key areas, health R&D and communicable disease control, in which the GPG concept is clearly relevant and considers the extent to which it has been applied. We point out that that, while there have been many new initiatives, it is not clear that additional resources from non-traditional sources have been forthcoming. Yet achieving this is, in effect, the entire purpose of applying the GPG concept in global health. Moreover, the proliferation of disease-specific programs associated with GPG reasoning has tended to promote vertical interventions at the expense of more general health sector strengthening. Third, we examine two major global health policy initiatives, the Global Fund against AIDS, Tuberculosis and Malaria (GFATM) and the bundling of long-standing international health goals in the form of Millennium Development Goals (MDG), asking how the GPG perspective has contributed to defining objectives and strategies. We conclude that both initiatives are best interpreted in the context of traditional development assistance and, one-world rhetoric aside, have little to do with the challenge posed by GPGs for health. The paper concludes by considering how the GPG concept can be more effectively used to promote global health.

Binding of Ni2+ ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution.

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups.

The cloning, expression, purification, crystallization and preliminary crystallographic analysis of mannosyl-3-phosphoglycerate phosphatase (MpgP) from T. thermophilus HB27 are reported. The stability of MpgP in solution was studied by size-exclusion chromatography and differential scanning fluorimetry assays.

Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallo­graphic studies. The crystals obtained belonged to the monoclinic space group P21, with unit-cell parameters a = 39.52, b = 70.68, c = 95.42 Å, β = 92.95°. Diffraction data were measured to 1.9 Å resolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.

A 500, 400 and 300 MHz proton NMR study of the reaction product of cis-Pt(NH3)2Cl2 or cis-[Pt(NH3)2 (H2O)2] (NO3)2 with the deoxydinucleotide d(GpG): cis-[Pt(NH3)2 d(GpG)] was carried out. Complete assignment of the proton resonances by decoupling experiments and computer simulation of the high field part of the spectrum yield proton-proton and proton-phosphorus coupling constants of high precision. Analysis of these coupling constants reveal a 100% N (C3'-endo) conformation for the deoxyribose ring at the 5'-terminal part of the chelated d(GpG) moiety. In contrast, the 3'-terminal -pG part of the molecule displays the normal behaviour for deoxyriboses: the sugar ring prefers to adopt an S (C2'-endo) conformation (about 70%). Extrapolating from this model compound, it is suggested that Pt chelation by a -dGpdG- sequence of DNA would require a S to N conformational change of one deoxyribose moiety as the main conformational alteration and lead to a kink in one strand of the double-helical structure of DNA.

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 Å, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.

Acetyl coenzyme A carboxylase (ACC) is a key enzyme providing a substrate for mycolic acid biosynthesis. Although in vitro studies have demonstrated that the protein encoded by accD6 (Rv2247) may be a functional carboxyltransferase subunit of ACC in Mycobacterium tuberculosis, the in vivo function and regulation of accD6 in slow- and fast-growing mycobacteria remain elusive. Here, directed mutagenesis demonstrated that although accD6 is essential for M. tuberculosis, it can be deleted in Mycobacterium smegmatis without affecting its cell envelope integrity. Moreover, we showed that although it is part of the type II fatty acid synthase operon, the accD6 gene of M. tuberculosis, but not that of M. smegmatis, possesses its own additional promoter (Pacc). The expression level of accD6Mtb placed only under the control of Pacc is 10-fold lower than that in wild-type M. tuberculosis but is sufficient to sustain cell viability. Importantly, this limited expression level affects growth, mycolic acid content, and cell morphology. These results provide the first in vivo evidence for AccD6 as a key player in the mycolate biosynthesis of M. tuberculosis, implicating AccD6 as the essential ACC subunit in pathogenic mycobacteria and an excellent target for new antitubercular compounds. Our findings also highlight important differences in the mechanism of acetyl carboxylation between pathogenic and nonpathogenic mycobacterial species.

The crystal structure of d(CATGGGCCCATG)2 shows unique stacking patterns of a stable B↔A-DNA intermediate. We evaluated intrinsic base stacking energies in this crystal structure using an ab initio quantum mechanical method. We found that all crystal base pair steps have stacking energies close to their values in the standard and crystal B-DNA geometries. Thus, naturally occurring stacking geometries were essentially isoenergetic while individual base pair steps differed substantially in the balance of intra-strand and inter-strand stacking terms. Also, relative dispersion, electrostatic and polarization contributions to the stability of different base pair steps were very sensitive to base composition and sequence context. A large stacking flexibility is most apparent for the CpA step, while the GpG step is characterized by weak intra-strand stacking. Hydration effects were estimated using the Langevin dipoles solvation model. These calculations showed that an aqueous environment efficiently compensates for electrostatic stacking contributions. Finally, we have carried out explicit solvent molecular dynamics simulation of the d(CATGGGCCCATG)2 duplex in water. Here the DNA conformation did not retain the initial crystal geometry, but moved from the B↔A intermediate towards the B-DNA structure. The base stacking energy improved in the course of this simulation. Our findings indicate that intrinsic base stacking interactions are not sufficient to stabilize the local conformational variations in crystals.

M. tuberculosis dihydrodipicolinate synthase, the enzyme that catalyzes the first unique reaction in the l-lysine biosynthesis pathway, has been cloned, expressed, purified and crystallized and the crystals have been characterized by X-ray diffraction.

Dihydrodipicolinate synthase from Mycobacterium tuberculosis (DHDPS, DapA, Rv2753c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in a monoclinic crystal form. Preliminary diffraction data analysis suggests the presence of two independent tetramers in the asymmetric unit in almost the same relative orientation.