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.     

Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus

For the first time, we demonstrate directly a stable complex between a bacterial DnaG (primase) and DnaB (helicase). Utilizing fragments of both proteins, we are able to dissect interactions within this complex and provide direct evidence that it is the C-terminal domain of primase that interacts with DnaB. Furthermore, this C-terminal domain is sufficient to induce maximal stimulation of the helicase and ATPase activities of DnaB. However, the region of DnaB that interacts with the C-terminal domain of primase appears to comprise a surface on DnaB that includes regions from both of the previously identified N- and C-terminal domains. Using a combination of biochemical and physical techniques, we show that the helicase-primase complex comprises one DnaB hexamer and either two or three molecules of DnaG. Our results show that in Bacillus stearothermophilus the helicase-primase interaction at the replication fork may not be transient, as was shown to be the case in Escherichia coli. Instead, primase appears to interact with the helicase forming a tighter complex with enhanced ATPase and helicase activities.     

DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings

Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase–primase (DnaB–DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB–DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested.     

Discovery of a compound which acts as a bacterial UMP kinase PyrH inhibitor

PyrH is a member of the UMP kinase family that catalyses the conversion of UMP to UDP, an essential step in the pyrimidine metabolic pathway in a variety of bacteria including those causing community-acquired respiratory tract infections (RTIs). In this study, we have developed a luminescence-based kinase assay of PyrH and evaluated the inhibitory activity of PYRH-1 (sodium {3-[4-tert-butyl-3-(9H-xanthen-9-ylacetylamino)phenyl]-1-cyclohexylmethylpropoxycarbonyloxy}acetate). PYRH-1 inhibits PyrH derived from both Streptococcus pneumoniae and Haemophilus influenzae with IC(50) (concentration of inhibitor giving a 50% decrease in enzyme activity) values of 48 and 75?μM, respectively, whose inhibitory activity against S.?pneumoniae PyrH was far higher compared with that of UTP (IC(50) =?710?μM), an allosteric PyrH inhibitor. The molecular interaction analysis by surface plasmon resonance suggested that PYRH-1 directly interacts with S.?pneumoniae PyrH at one-to-one molar ratio. Finally, PYRH-1 was shown to have antimicrobial activity against several different bacteria causing RTIs, such as S.?pneumoniae, Staphylococcus aureus, H.?influenzae (acrA knockout strain), suggesting that PYRH-1 is a prototype chemical compound that can be harnessed as an antimicrobial drug with a novel mode of action by targeting bacterial PyrH.     

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.     

Crystal structure of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture

The bifunctional bacterial enzyme N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against beta-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 A, respectively. The S. pneumoniae GlmU molecule is organized in two separate domains connected via a long alpha-helical linker and associates as a trimer, with the 50-A-long left-handed beta-helix (LbetaH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LbetaH core and exchanged with the beta-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LbetaH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.     

Identification and validation of bioactive small molecule target through phenotypic screening

For effective bioactive small molecule discovery and development into new therapeutic drug, a systematic screening and target protein identification is required. Different from the conventional screening system, herein phenotypic screening in combination with multi-omics-based target identification and validation (MOTIV) is introduced. First, phenotypic screening provides visual effect of bioactive small molecules in the cell or organism level. It is important to know the effect on the cell or organism level since small molecules affect not only a single target but the entire cellular mechanism within a cell or organism. Secondly, MOTIV provides systemic approach to discover the target protein of bioactive small molecule. With the chemical genomics and proteomics approach of target identification methods, various target protein candidates are identified. Then network analysis and validations of these candidates result in identifying the biologically relevant target protein and cellular mechanism. Overall, the combination of phenotypic screening and MOTIV will provide an effective approach to discover new bioactive small molecules and their target protein and mechanism identification.     

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.     

Triclosan-lysozyme complex as novel antimicrobial macromolecule: a new potential of lysozyme as phenolic drug-targeting molecule

A novel anti-infection strategy to alleviate antibiotic-resistance problem and non-specific toxicity associated with chemotherapy is explored in this study. It is based on utilizing a bacteriolytic enzyme (lysozyme) as a carrier to allow specific targeting of a potential phenolic antimicrobial drug (triclosan) to microbial cells. Lysozyme (LZ) was complexed, via electrostatic and hydrophobic condensation at alkaline pH, to various degrees with triclosan (TCS), a negatively charged phenolic antimicrobial that inhibits bacterial fatty acid synthesis. Fluorescence and absorbance spectra analysis revealed non-covalent association of TCS with the aromatic residues at the interior of LZ molecule. The conjugation greatly promoted the lytic activity of LZ as the degree of TCS derivatization increased. The complexation with LZ turned TCS into completely soluble in aqueous solution. TCS-LZ complexes showed significantly enhanced bactericidal activity against several strains of Gram-positive and Gram-negative bacteria compared to the activity of TCS or LZ alone when tested at the same molar basis. Strikingly, TCS-LZ complex, but not LZ or TCS alone, exhibited unique specificity to scavenge superoxide radicals, generated by the natural xanthine/xanthine oxidase coupling system, without affecting the catalytic function of oxidase. This finding is the first to describe that the membrane disrupting function of lysozyme can be utilized to specifically target antimicrobial drug(s) to pathogen cells and heralding a fascinating opportunity for the potential candidacy of TCS-LZ as novel antimicrobial strategy for human therapy.     

GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae.

Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine-6-phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to p-chloromercuribenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glucosamine 6-phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxicity in eukaryotic cells.     

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.     

Inhibitors of fatty acid synthesis as antimicrobial chemotherapeutics

Fatty acid biosynthesis is an emerging target for the development of novel antibacterial chemotherapeutics. The dissociated bacterial system is substantially different from the large, multifunctional protein of mammals, and many possibilities exist for type-selective drugs. Several compounds, both synthetic and natural, target bacterial fatty acid synthesis. Three compounds target the FabI enoyl-ACP reductase step; isoniazid, a clinically used antituberculosis drug, triclosan, a widely used consumer antimicrobial, and diazaborines. In addition, cerulenin and thiolactomycin, two fungal products, inhibit the FabH, FabB and FabF condensation enzymes. Finally, the synthetic reaction intermediates BP1 and decynoyl- N-acetyl cysteamine inhibit the acetyl-CoA carboxylase and dehydratase isomerase steps, respectively. The mechanisms of action of these compounds, as well as the potential development of new drugs targeted against this pathway, are discussed.     

Bacterial virulence as a target for antimicrobial chemotherapy

As bacterial resistance to currently used antibiotics increases, so too must efforts to identify novel agents and strategies for the prevention and treatment of bacterial infection. In the past, antimicrobial drug discovery efforts have focused on eradicating infection by either cidal or static agents, resulting in clearance of the bacterium from the infected host. To this end, drug discovery targets have been those proteins or processes essential for bacterial cell viability. However, inhibition of the interaction between the bacterium and its host may also be a target. During establishment of an infection, pathogenic bacteria use carefully regulated pathways of conditional gene expression to transition from a free-living form to one that must adapt to the host milieu. This transition requires the regulated production of both extracellular and cell-surface molecules, often termed virulence factors. As the biological imperatives of the invading organism change during the course of an infection, the expression of these factors is altered in response to environmental cues. These may be changes in the host environment, for example, pH, metabolites, metal ions, osmolarity, and temperature. Alternatively, effector molecules produced by the bacterium to sense changing cell density can also lead to changes in virulence gene expression. Although the mechanisms of pathogenesis among different bacteria vary, the principles of virulence are generally conserved. Bacterial virulence may therefore offer unique opportunities to inhibit the establishment of infection or alter its course as a method of antimicrobial chemotherapy.     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

抗菌肽作用机制的研究进展

抗菌肽是一类来源于多种生物、能有效杀灭病原体的小分子多肽,具有活性谱广、作用强且迅速、不易产生耐药等众多优点.作为新一代抗感染候选药物,抗菌肽的作用机制还未完全清楚,但目前有两种观点已得到公认,即胞膜渗透作用破坏胞膜结构完整性和作用于胞内不同靶点干扰细菌生长及代谢平衡.本文主要就抗菌肽理化性质、二级结构、作用机制以及后两者间的关系做一总结,以便更好的理解抗菌肽的构效关系,为合理设计抗菌肽提供理论基础.     

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily.

N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide-activated UDP-GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 A resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long alpha-helical arm: (i) an N-terminal domain which resembles the dinucleotide-binding Rossmann fold; and (ii) a C-terminal domain which adopts a left-handed parallel beta-helix structure (LbetaH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LbetaH domains, the long alpha-helical linkers being seated on top of the arrangement and the N-terminal domains projected away from the 3-fold axis. In addition, the 2.3 A resolution structure of the GlmU-UDP-GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three-dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP-sugar bacterial pyrophosphorylases.     

Comparative antibacterial activity of a novel semisynthetic antibiotic: etimicin sulphate and other aminoglycosides

Etimicin is a novel fourth generation semisynthetic aminoglycoside. It has good antimicrobial activity against both gram-positive and gram-negative bacterial infections and also against aminoglycoside resistant strains. In the present study, in vitro antibacterial activity of etimicin was determined by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill curve tests against type strains and 407 clinical isolates (obtained in a surviellance study), in comparison to other aminoglycosides. Test results revealed that etimicin has potential antimicrobial activity and MIC, MBC values for etimicin were low compared to other aminoglycosides. In MBC test etimicin has exhibited potential bactericidal effect ranging from 0.25 to 2?mg/L. The time kill-curve study further demonstrated the rapid, concentration dependent killing and comparative study showed etimicin to exhibit long and effective bactericidal activity over amikacin. The interesting fact is that most of the tested aminoglycoside resistant clinical isolates were susceptible to etimicin. In view of its potent in vitro antibacterial activity and efficacy profiles, it can be concluded that etimicin can be a potent injectable agent for the treatment of severe bacterial infections.     

Isolation and identification of three bactericidal domains in the bovine alpha-lactalbumin molecule

Proteolytic digestion of alpha-lactalbumin by pepsin, trypsin and chymotrypsin yielded three polypeptide fragments with bactericidal properties. Two fragments were obtained from the tryptic digestion. One was a pentapeptide with the sequence EQLTK (residues 1-5) and the other, GYGGVSLPEWVCTTF ALCSEK (residues (17-31)S-S(109-114)), was composed of two polypeptide chains held together by a disulfide bridge. Fragmentation of alpha-lactalbumin by chymotrypsin yielded CKDDQNPH ISCDKF (residues (61-68)S-S(75-80)), also a polypeptide composed of two polypeptide chains held together by a disulfide bridge. The three polypeptides were synthesized and found to exert antimicrobial activities. The polypeptides were mostly active against Gram-positive bacteria. Gram-negative bacteria were only poorly susceptible to the bactericidal action of the polypeptides. GYGGVSLPEWVCTTF ALCSEK was most, EQLTK least bactericidal. Replacement of leucine (23) with isoleucine, having a similar chemical structure but higher hydrophobicity, in the sequence GYGGVSLPEWVCTTF ALCSEK significantly reduced the bactericidal capacity of the polypeptide. Digestion of alpha-lactalbumin by pepsin yielded several polypeptide fragments without antibacterial activity. alpha-Lactalbumin in contrast to its polypeptide fragments was not bactericidal against all the bacterial strains tested. Our results suggest a possible antimicrobial function of alpha-lactalbumin after its partial digestion by endopeptidases.     

Bioenergetic and proteomic profiling to screen small molecule inhibitors that target cancer metabolisms

Cancer cells can reprogram their metabolic machinery to survive. This altered metabolism, which is distinct from the metabolism of normal cells, is thought to be a possible target for the development of new cancer therapies. In this study, we constructed a screening system that focuses on bioenergetic profiles (specifically oxygen consumption rate and extracellular acidification rate) and characteristic proteomic changes. Thus, small molecules that target cancer-specific metabolism were investigated. We screened the chemical library of RIKEN Natural Products Depository (NPDepo) and found that unantimycin A, which was recently isolated from the fraction library of microbial metabolites, and NPL40330, which is derived from a chemical library, inhibit mitochondrial respiration. Furthermore, we developed an in vitro reconstitution assay method for mitochondrial electron transport chain using semi-intact cells with specific substrates for each complex of the mitochondrial electron transport chain. Our findings revealed that NPL40330 and unantimycin A target mitochondrial complexes I and III, respectively.     

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.