Identification of amino acids involved in catalytic process of M. tuberculosis GlmU acetyltransferase

M. tuberculosis GlmU is a bifunctional enzyme with acetyltransferase activity in C-terminus and uridyltransferase activity in N-terminus, and it is involved in the biosynthesis of glycosyl donor UDP-N-acetylglucosamine (UDP-GlcNAc). The crystal structure of M. tuberculosis GlmU clearly determines the active site and catalytic mechanism of GlmU uridyltransferase domain but not succeed in GlmU acetyltransferase domain. Sequence comparison analysis revealed highly conserved amino acid residues in the C-terminus between M. tuberculosis GlmU and GlmU enzymes from other bacteria. To find the essential amino acids related to M. tuberculosis GlmU acetyltransferase activity, we substituted 10 conserved amino acids in the acetyltransferase domain of M. tuberculosis GlmU by site-directed mutagenesis. All the mutant GlmU proteins were largely expressed in soluble and purified by affinity chromatography. Enzyme assays showed that K362A, H374A, Y398A and W460A mutants abolished more than 90?% activity of M. tuberculosis GlmU acetyltransferase and totally lost the affinity with two substrates, suggesting the potential substrate-binding functions. However, K403A, S416A, N456A and E458A mutants exhibited decreased GlmU acetyltransferase activity and lower kinetic parameters, probably responsible for substrate releasing by conformation shifting.     

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmU^Mtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmU^Mtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmU^Mtb. In this S_N2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmU^Mtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmU^Mtb, which may be exploited for the development of inhibitors specific to GlmU.     

In vitro validation of acetyltransferase activity of GlmU as an antibacterial target in Haemophilus influenzae

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.     

Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products

The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO(4) to form GlcNAc-1-PO(4) and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO(4), and desulpho-CoA/GlcNAc-1-PO(4). These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO(4) is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.     

Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth

The bifunctional N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) enzyme catalyzes both the acetylation of glucosamine 1-phosphate and the uridylation of N-acetylglucosamine 1-phosphate, two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis in bacteria. In our previous work describing its initial characterization in Escherichia coli, we proposed that the 456-amino acid (50.1 kDa) protein might possess separate uridyltransferase (N-terminal) and acetyltransferase (C-terminal) domains. In the present study, we confirm this hypothesis by expression of the two independently folding and functional domains. A fragment containing the N-terminal 331 amino acids (Tr331, 37.1 kDa) has uridyltransferase activity only, with steady-state kinetic parameters similar to the full-length protein. Further deletion of 80 amino acid residues at the C terminus results in a 250-amino acid fragment (28.6 kDa) still exhibiting significant uridyltransferase activity. Conversely, a fragment containing the 233 C-terminal amino acids (24.7 kDa) exhibits acetyltransferase activity exclusively. None of these individual domains could complement a chromosomal glmU mutation, indicating that each of the two activities is essential for cell viability. Analysis of truncated GlmU proteins by gel filtration further localizes regions of the protein involved in its trimeric organization. Interestingly, overproduction of the truncated Tr331 protein in a wild-type strain results in a rapid depletion of endogenous acetyltransferase activity, an arrest of peptidoglycan synthesis and cell lysis. It is shown that the acetyltransferase activity of the full-length protein is abolished once trapped within heterotrimers formed in presence of the truncated protein, suggesting that this enzyme activity absolutely requires a trimeric organization and that the catalytic site involves regions of contact between adjacent monomers. Data are discussed in connection with the recently obtained crystal structure of the truncated Tr331 protein.     

Kinetic properties of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> bifunctional GlmU

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg²⁺, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.     

Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis.

The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D. Mengin-Lecreulx and J. van Heijenoort, J. Bacteriol. 175:6150-6157, 1993). Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided. Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity. In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site. The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway. The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria.     

An aminoquinazoline inhibitor of the essential bacterial cell wall synthetic enzyme GlmU has a unique non-protein-kinase-like binding mode

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 ?. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P (N-acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure-activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery.     

Characterization of substrate binding and catalysis in the potential antibacterial target N-acetylglucosamine-1-phosphate uridyltransferase (GlmU)

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the first step in peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. The products of the GlmU reaction are essential for bacterial survival, making this enzyme an attractive target for antibiotic drug discovery. A series of Haemophilus influenzae GlmU (hiGlmU) structures were determined by X-ray crystallography in order to provide structural and functional insights into GlmU activity and inhibition. The information derived from these structures was combined with biochemical characterization of the K25A, Q76A, D105A, Y103A, V223A, and E224A hiGlmU mutants in order to map these active-site residues to catalytic activity of the enzyme and refine the mechanistic model of the GlmU uridyltransferase reaction. These studies suggest that GlmU activity follows a sequential substrate-binding order that begins with UTP binding noncovalently to the GlmU enzyme. The uridyltransferase active site then remains in an open apo-like conformation until N-acetylglucosamine-1-phosphate (GlcNAc-1-P) binds and induces a conformational change at the GlcNAc-binding subsite. Following the binding of GlcNAc-1-P to the UTP-charged uridyltransferase active site, the non-esterified oxygen of GlcNAc-1-P performs a nucleophilic attack on the alpha-phosphate group of UTP. The new data strongly suggest that the mechanism of phosphotransfer in the uridyltransferase reaction in GlmU is primarily through an associative mechanism with a pentavalent phosphate intermediate and an inversion of stereochemistry. Finally, the structural and biochemical characterization of the uridyltransferase active site and catalytic mechanism described herein provides a basis for the structure-guided design of novel antibacterial agents targeting GlmU activity.     

Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase, GlmU, in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg(2+) at 1.96 A resolution

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 A resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg(2+) at 1.96 A resolution. The protein structure consists of an N-terminal domain with an alpha/beta-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed beta-sheet helix (LbetaH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed beta-sheet helix domain. In the crystal, S. pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed beta-sheet helix domains. UDP-N-acetylglucosamine and Mg(2+) are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg(2+) and the side-chain of Lys22.     

Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization

In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in ≥94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY(G153D) had 10-fold higher affinity for GTP than MjCobY(WT) but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY(G153D) in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).     

Systematic study on the broad nucleotide triphosphate specificity of the pyrophosphorylase domain of the N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Escherichia coli K12 is a bifunctional enzyme that catalyzes both the acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthetic pathway. In this study, we report the broad substrate specificity of the pyrophosphorylase domain of GlmU during its uridyltransfer reaction and the substrate priority is ranked in the following order: UTP > dUTP > dTTP >> CTP > dATP/dm⁶ ATP. This pyrophosphorylase domain of GlmU is also a tool to synthesize UDP-GlcNAc analogs, two examples of which were synthesized herein in multiple mg scale in vitro.     

Self-association studies of the bifunctional N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli

The N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a key bifunctional enzyme in the biosynthesis of UDP-GlcNAc, a precursor in the synthesis of cell wall peptidoglycan. Crystal structures of the enzyme from different bacterial strains showed that the polypeptide forms a trimer through a unique parallel left-handed beta helix domain. Here, we show that the GlmU enzyme from Escherichia coli forms a hexamer in solution. Sedimentation equilibrium analytical ultracentrifugation demonstrated that the enzyme is in a trimer/hexamer equilibrium. Small-angle X-ray scattering studies were performed to determine the structure of the hexameric assembly and showed that two trimers assemble through their N-terminal domains. The interaction is mediated by a loop that undergoes a large conformational change in the uridyl transferase reaction, a feature that may affect the enzymatic activity of GlmU.     

Thermodynamic and kinetic destabilization of triosephosphate isomerase resulting from the mutation of conserved and non-conserved cysteines

Several variants of Saccharomyces cerevisiae triosephosphate isomerase (yTIM) were studied to determine how mutations of conserved and non-conserved Cys residues affect the enzyme. Wild-type yTIM has two buried free cysteines: Cys 41 (non-conserved) and the invariant Cys 126. Single-site mutants, containing substitutions of these cysteines with Ala, Val, or Ser (the three most conservative changes for a buried Cys, according to substitution matrices), were examined for stability and enzymatic activity. Neither of the Cys residues was found to be essential for enzyme catalysis. Determination of the global stability of the mutants indicated that, regardless of which Cys was substituted, individual Cys→Ala and Cys→Val mutations, as well as the C41S substitution, all decrease the unfolding free energy of the dimeric protein by less than 23 kJ mol(-1) (at 37 °C, pH 7.4), as compared to the wild-type enzyme. In contrast, a substantially larger destabilization (37 kJ mol(-1)) was found in the C126S mutant. These results suggest that, with the exception of C126S, all of these mutations can be regarded as neutral (i.e., mutations that do not impair the reproductive success of the organism). Accordingly, Cys 126 has remained invariant across evolution because its neutral substitutions by Ala or Val would require a highly unlikely, concerted double mutation at any of the Cys codons. Furthermore, detrimental effects to a cell expressing the C126S TIM mutant more likely arise from the high unfolding rate of this enzyme.     

Inhibitors of the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridylyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). Part 2: Optimization of physical properties leading to antibacterial aryl sulfonamides

A previously described aryl sulfonamide series, originally found through HTS, targets GlmU, a bifunctional essential enzyme involved in bacterial cell wall synthesis. Using structure-guided design, the potency of enzyme inhibition was increased in multiple isozymes from different bacterial species. Unsuitable physical properties (low Log D and high molecular weight) of those compounds prevented them from entering the cytoplasm of bacteria and inhibiting cell growth. Further modifications described herein led to compounds that possessed antibacterial activity, which was shown to occur through inhibition of GlmU. The left-hand side amide and the right-hand side sulfonamides were modified such that enzyme inhibitory activity was maintained (IC₅₀ <0.1 μM against GlmU isozymes from Gram-negative organisms), and the lipophilicity was increased giving compounds with Log D ?1 to 3. Antibacterial activity in an efflux-pump deficient mutant of Haemophilus influenzae resulted for compounds such as 13.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

Influence of multiple cysteines on human 3-hydroxy-3-methylglutaryl-CoA lyase activity and formation of inter-subunit adducts

Human 3-hydroxy-3-methylglutaryl-CoA lyase catalyzes formation of acetyl-CoA and acetoacetate in a reaction that requires divalent cation and is stimulated by sulfhydryl protective reagents. The enzyme is a homodimer and inter-subunit adducts form in the absence of reducing agents or upon treatment with cysteine selective crosslinking agents. To address the influence of cysteines on enzyme activity and formation of inter-subunit and intra-subunit adducts, single serine substitutions have been engineered for each enzyme cysteine. Enzyme activity varies for each cysteine→serine mutant protein and different mutations have widely different effects on recovery of activity upon DTT treatment of non-reduced enzyme. These levels of enzyme activity do not strongly correlate with formation of inter-subunit adducts by these HMGCL mutants. C170S, C266S, and C323S proteins do not form inter-subunit disulfide adducts but such an adduct is restored in the C170S/C174S double mutant. Coexpression of HMGCL proteins encoded by C266S and C323S expression plasmids supports formation of a C266S/C323S heterodimer which does form a covalent inter-subunit adduct. These observations are interpreted in the context of competition between cysteines in formation of intra-subunit and inter-subunit heterodisulfide adducts.     

Kinetic Modelling of GlmU Reactions - Prioritization of Reaction for Therapeutic Application

Mycobacterium tuberculosis(Mtu), a successful pathogen, has developed resistance against the existing anti-tubercular drugs necessitating discovery of drugs with novel action. Enzymes involved in peptidoglycan biosynthesis are attractive targets for antibacterial drug discovery. The bifunctional enzyme mycobacterial GlmU (Glucosamine 1-phosphate N-acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase) has been a target enzyme for drug discovery. Its C- and N- terminal domains catalyze acetyltransferase (rxn-1) and uridyltransferase (rxn-2) activities respectively and the final product is involved in peptidoglycan synthesis. However, the bifunctional nature of GlmU poses difficulty in deciding which function to be intervened for therapeutic advantage. Genetic analysis showed this as an essential gene but it is still unclear whether any one or both of the activities are critical for cell survival. Often enzymatic activity with suitable high-throughput assay is chosen for random screening, which may not be the appropriate biological function inhibited for maximal effect. Prediction of rate-limiting function by dynamic network analysis of reactions could be an option to identify the appropriate function. With a view to provide insights into biochemical assays with appropriate activity for inhibitor screening, kinetic modelling studies on GlmU were undertaken. Kinetic model of Mtu GlmU-catalyzed reactions was built based on the available kinetic data on Mtu and deduction from Escherichia coli data. Several model variants were constructed including coupled/decoupled, varying metabolite concentrations and presence/absence of product inhibitions. This study demonstrates that in coupled model at low metabolite concentrations, inhibition of either of the GlmU reactions cause significant decrement in the overall GlmU rate. However at higher metabolite concentrations, rxn-2 showed higher decrement. Moreover, with available intracellular concentration of the metabolites and in vivo variant of model, uncompetitive inhibition of rxn-2 caused highest decrement. Thus, at physiologically relevant metabolite concentrations, targeting uridyltranferase activity of Mtu GlmU would be a better choice for therapeutic intervention.     

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.     

Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites

N-Acetylglucosamine-1-PO(4) uridyltransferase (GlmU) is a trimeric bifunctional enzyme that catalyzes the last two sequential reactions in the de novo biosynthetic pathway for UDP-GlcNAc. The X-ray crystal structure of Escherichia coli GlmU in complex with UDP-GlcNAc and CoA has been determined to 2.1 A resolution and reveals a two-domain architecture that is responsible for these two reactions. The C-terminal domain is responsible for the CoA-dependent acetylation of Glc-1-PO(4) to GlcNAc-1-PO(4) and displays the longest left-handed parallel beta-helix observed to date. The acetyltransferase active site defined by the binding site for CoA makes use of residues from all three subunits and is positioned beneath an open cavity large enough to accommodate the Glc-1-PO(4) acetyl acceptor. The N-terminal domain catalyzes uridyl transfer from UTP to GlcNAc-1-PO(4) to form the final products UDP-GlcNAc and pyrophosphate. This domain is composed of a central seven-stranded beta-sheet surrounded by six alpha-helices in a Rossmann fold-like topology. A Co(2+) ion binds to just one of the two independent pyrophosphorylase active sites present in the crystals studied here, each of which nonetheless binds UDP-GlcNAc. The conformational changes of the enzyme and sugar nucleotide that accompany metal binding may provide a window into the structural dynamics that accompany catalysis.