Uridyltransferase Activity Research Articles

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.

Disturbed Galactose Metabolism in Elderly and Diabetic Humans Is Associated with Cataract Formation

Lactose consumption has been associated with a high incidence of cataract in northern Indian and southern Italian populations. Galactose absorbed after hydrolysis of lactose from milk in individuals with normal lactase activity is considered responsible. However, lactase-deficient subjects who often avoid drinking milk are able to digest lactose and absorb free galactose in fermented milk and yogurt. This study was conducted to evaluate the relationships between milk and yogurt consumption, galactose metabolism and cataract risk. Milk ingestion was dose-related with cataract risk in lactose digesters (particularly in diabetics) but not in lactose maldigesters. Conversely, yogurt intake had a protective dose-effect on cataract formation for the whole population. Maximal galactose concentrations after an oral galactose test increased exponentially with age. Red blood cell galactokinase activity was significantly lower in elderly subjects (> 60 y) than in young individuals (P < 0.05), and galactose-1-phosphate uridyl-transferase activity was significantly lower in institutionalized subjects and in home-living elderly with cataract than in healthy elderly subjects (P < 0.05). We conclude that the cataractogenic action of milk lactose is dependent on the disturbance of galactose metabolism in elderly subjects and that yogurt is not cataractogenic, although the mechanism of the protective effect of yogurt remains unknown.

In vitro particle-associated uridyltransferase activity of the rotavirus VP1 polymerase

Rotaviruses are 11-segmented, double-stranded RNA (dsRNA) viruses with a unique intra-particle RNA synthesis mechanism. During genome replication, the RNA-dependent RNA polymerase (VP1) performs minus-strand RNA (-ssRNA) synthesis on positive-strand RNA (+ssRNA) templates to create dsRNA segments. Recombinant VP1 catalyzes -ssRNA synthesis using substrate NTPs in vitro, but only when the VP2 core shell protein or virus-like particles made of VP2 and VP6 (2/6-VLPs) are included in the reaction. The dsRNA product can be labeled using [α32P]-UTP and separated from the input +ssRNA template by polyacrylamide gel electrophoresis. Here, we report the generation of [α32P]-labeled rotavirus +ssRNA templates in reactions that lacked non-radiolabeled NTPs but contained catalytically-active VP1, 2/6-VLPs, and [α32P]-UTP. Non-radiolabeled UTP competed with [α32P]-UTP to decrease product levels, whereas CTP and GTP had little effect. Interesting, ATP stimulated [α32P]-labeled product production. These results suggest that rotavirus VP1 transferred [α32P]-UMP onto viral + ssRNA in vitro via a particle-associated uridyltransferase activity.

Identification of Mtb GlmU Uridyltransferase Domain Inhibitors by Ligand-Based and Structure-Based Drug Design Approaches.

Targeting enzymes that play a role in the biosynthesis of the bacterial cell wall has long been a strategy for antibacterial discovery. In particular, the cell wall of Mycobacterium tuberculosis (Mtb) is a complex of three layers, one of which is Peptidoglycan, an essential component providing rigidity and strength. UDP-GlcNAc, a precursor for the synthesis of peptidoglycan, is formed by GlmU, a bi-functional enzyme. Inhibiting GlmU Uridyltransferase activity has been proven to be an effective anti-bacterial, but its similarity with human enzymes has been a deterrent to drug development. To develop Mtb selective hits, the Mtb GlmU substrate binding pocket was compared with structurally similar human enzymes to identify selectivity determining factors. Substrate binding pockets and conformational changes upon substrate binding were analyzed and MD simulations with substrates were performed to quantify crucial interactions to develop critical pharmacophore features. Thereafter, two strategies were applied to propose potent and selective bacterial GlmU Uridyltransferase domain inhibitors: (i) optimization of existing inhibitors, and (ii) identification by virtual screening. The binding modes of hits identified from virtual screening and ligand growing approaches were evaluated further for their ability to retain stable contacts within the pocket during 20 ns MD simulations. Hits that are predicted to be more potent than existing inhibitors and selective against human homologues could be of great interest for rejuvenating drug discovery efforts towards targeting the Mtb cell wall for antibacterial discovery.

Assessment of galactose-1-phosphate uridyltransferase activity in cells and tissues.

Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring galacose-1-phosphate uridyltransferase activity in cell culture and animal tissues.

Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts it's in vivo function and the survival of pathogen.

N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmUMt) has features, such as a C-terminal extension, that are not present in GlmUorthologs from other bacteria. Here, we set out to determine the uniqueness of GlmUMt by performing in vivo complementation experiments using RvΔglmU mutant. We find that any deletion of the carboxy-terminal extension region of GlmUMt abolishes its ability to complement the function of GlmUMt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmUMt. Furthermore, the co-expression of GlmUMt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmUMt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmUMt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmUMt participates in unique interactions essential for its in vivo function.

Action of Dicumarol on Glucosamine-1-Phosphate Acetyltransferase of GlmU and Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of most pathogenic microorganisms in the world. Previously, the bifunctional enzyme GlmU with glucosamine-1-phosphate acetyltransferase activity and N-acetylglucosamine-1-phosphate uridyltransferase activity has been suggested as a potential drug target; therefore, discovering compounds targeting GlmU acetyltransferase is necessary. The natural products were tested for inhibition of GlmU acetyltransferase activity. We found that dicumarol exhibited inhibitory effects on GlmU acetyltransferase, with a concentration achieving a 50% inhibition (IC50) value of 4.608 μg/ml (13.7 μM). The inhibition kinetics indicated that dicumarol uncompetitively inhibited acetyl CoA and showed mixed-type inhibition for glucosamine-1-phosphate (GlcN-1-P). The activity of dicumarol against M. tuberculosis H37Ra was evaluated with a minimum inhibitory concentration (MIC) value of 6.25 μg/ml (18.55 μM) in the Alamar blue assay. Dicumarol also exhibited inhibitory effects on several clinically sensitive M. tuberculosis strains and drug-resistant strains, with a range of MIC value of 6.25 to >100 μg/ml. Dicumarol increased the sensitivity of anti-tuberculosis drugs (isoniazid and rifampicin) when dicumarol was present at a low concentration. The transcriptome and proteome data of M. tuberculosis H37Ra treated by dicumarol showed that the affected genes were associated with cell wall synthesis, DNA damage and repair, metabolic processes, and signal transduction. These results provided the mechanism of dicumarol inhibition against GlmU acetyltransferase and M. tuberculosis and also suggested that dicumarol is a potential candidate for TB treatment.

Including Classical Galactosaemia in the Expanded Newborn Screening Panel Using Tandem Mass Spectrometry for Galactose-1-Phosphate

Galactosaemia has been included in various newborn screening programs since 1963. Several methods are used for screening; however, the predominant methods used today are based on the determination of either galactose-1-phosphate uridyltransferase (GALT) activity or the concentration of total galactose. These methods cannot be multiplexed and therefore require one full punch per sample. Since the introduction of mass spectrometry in newborn screening, many diseases have been included in newborn screening programs. Here, we present a method for including classical galactosaemia in an expanded newborn screening panel based on the specific determination of galactose-1-phosphate by tandem mass spectrometry. The existing workflow only needs minor adjustments, and it can be run on the tandem mass spectrometers in routine use. Furthermore, compared to the currently used methods, this novel method has a superior screening performance, producing significantly fewer false positive results. We present data from 5500 routine newborn screening samples from the Danish Neonatal Screening Biobank. The cohort was enriched by including 14 confirmed galactosaemia positive samples and 10 samples positive for other metabolic disorders diagnosed through the Danish newborn screening program. All galactosaemia positive samples were identified by the method with no false positives. Furthermore, the screening performance for other metabolic disorders was unaffected.

Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity.IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.

Galactose-1-Phosphate Uridyltransferase Activities in Different Genotypes: A Retrospective Analysis of 927 Samples.

Classic galactosemia is an inherited disorder of galactose metabolism caused by the impaired activity of galactose-1-phosphate uridyltransferase (GALT). Untreated galactosemia is life-threatening; however, early dietary intervention prevents mortality and reduces morbidity associated with this disease. The diagnosis of galactosemia includes the measurement of GALT activity in red blood cells (RBC) and GALT gene analysis. In this study, we evaluate GALT activity in different genotypes using the results of combined biochemical and molecular testing in 927 samples. GALT activity in RBC was measured by LC-MS/MS. The analysis of the GALT gene was performed by targeted gene analysis and/or full gene sequencing. Samples were assigned based on the presence of pathogenic (G) or Duarte 2 (D) variants, or their absence (Neg), to G/G, D/G, G/Neg, D/D, D/Neg, and Neg/Neg genotypes. Finite mixture models were applied to investigate distributions of GALT activities in these genotypes. The reference ranges were determined using the central 95% of values of GALT activities. The ranges of GALT activity in G/G, D/G, G/Neg, D/D, D/Neg, and Neg/Neg genotypes are 0.0 to 0.7 μmol·h-1 gHb-1, 3.1 to 7.8 μmol·h-1 gHb-1, 6.5 to 16.2 μmol·h-1 gHb-1, 6.4 to 16.5 μmol·h-1 gHb-1, 12.0 to 24.0 μmol·h-1 gHb-1, and 19.4 to 33.4 μmol·h-1 gHb-1, respectively. The GALT activity ranges established in this study are in agreement with the expected impact of the genotype on the enzymatic activity. Molecular findings should be interpreted in view of biochemical results to confirm genotype-phenotype correlation.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

Most organisms, from Bacteria to Eukarya, synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP-N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways.IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.

Mechanism and Potential Inhibitors of GlmU: A Novel Target for Antimicrobial Drug Discovery.

Multidrug resistant Gram negative pathogens pose a persistent threat to the health care system and require investigation of new targets and molecules for the development of antibiotics to treat infections caused by MDR bacterial pathogens. It is essential to work on multidisciplinary approaches and diverse strategies for developing new compounds acting on novel antibacterial targets. N-acetylglucosamine-1-phosphateuridyltransferase/ glucosamine-1-phosphate-acetyltransferase (GlmU) is one such target which is involved in the synthesis of both peptidoglycan and Lipopolysaccharide in Gram negative and Gram positive bacteria making GlmU an attractive target for developing antibacterials. GlmU, as revealed by X- ray crystallographic studies, is made up of two domains connected by α helical arm; the N-terminal uridyltransferase domain resembles a dinucleotide Rossmann fold and the C- terminal acetyltransferase domain adopts a left handed parallel β helix structure. The GlmU molecules are arranged in a trimeric array, the acetyltransferase active site being formed at the junction of adjacent LβH domain. Many potent inhibitors of both the acetyltransferase and uridyltransferase activity of GlmU have been identified. Inhibitors of the acetyltransferase activity of GlmU include nonspecific thiol reactive agents, 2-phenylbenzofurans, arylamines and arylsulfonamides. A aminopiperidine based inhibitor has also been reported to inhibit uridyltransferase activity of hiGlmU. The present review provides an insight of the structure of GlmU and its reported inhibitors which make GlmU a potential target for drug designing. The GlmU is a promising target for drug discovery against Gram negative pathogens and future studies should focus on GlmU for the development of more potent compounds for treating Gram negative infections.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent kcat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.

Probing the roles of conserved residues in uridyltransferase domain of Escherichia coli K12 GlmU by site-directed mutagenesis

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme that catalyzes both acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthesis pathway. Our previous study demonstrated that the uridyltransferase domain of GlmU (tGlmU) exhibited a flexible substrate specificity, which could be further applied in unnatural sugar nucleotides preparation. However, the structural basis of tolerating variant substrates is still not clear. Herein, we further investigated the roles of several highly conserved amino acid residues involved in substrate binding and recognition by structure- and sequence-guided site-directed mutagenesis. Out of total 16 mutants designed, tGlmU Q76E mutant which had a novel catalytic activity to convert CTP and GlcNAc-1P into unnatural sugar nucleotide CDP-GlcNAc was identified. Furthermore, tGlmU Y103F and N169R mutants were also investigated to have enhanced uridyltransferase activities compared with wide-type tGlmU.

Novel lead compound optimization and synthesized based on the target structure of Xanthomonas oryzae pv. oryzae GlmU

Bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases of rice worldwide. N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) was an attractive target for the development of antimicrobial agents. To develop novel, more potent and even more selective inhibitors of the uridyltransferase activity of Xanthomonas oryzae pv. oryzae GlmU (Xo-GlmU), three types of novel target compounds were optimized and synthesized based on the Xo-GlmU structure in this study. The biological testing results showed that all of the target compounds displayed the higher inhibition than the lead compound with the IC50 values in the 10.82–23.31 µM range, and the inhibition rates were increased by 30%–67%. The binding mode and the possible inhibitory mechanism of the target compounds in the active site were also analyzed by the molecular docking based on the uridyltransferase active site of Xo-GlmU.

Purification and biochemical characterisation of GlmU from Yersinia pestis.

Antibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents.

Galactosemia como causa de ictericia neonatal

Jaundice is a commonly occurring problem in hospitals' neonatal units. This report describes a newborn with hypefbilirrubinaemia; the clinical picture was initially dominated by the unconjugated bilirubin fraction and later by anaemia, hepatomegaly, increased conjugated bilirubin fraction, impaired liver function, renal tubular dysfunction, cataracts and injury to CNS white matter. Various causes of direct hyperbilirrubinaemia in the newborn were ruled out. Thin-layer chromatography revealed urine-reducing substances (i.e. galactose). A marked reduction in galactose uridyl transferaseactivity was documented, thereby confirming a diagnosis of galactosaemia. Breastfeedingwas discontinued and soy milk started which led to the symptoms becoming resolved.

Novel GALT variations and mutation spectrum in the Korean population with decreased galactose-1-phosphate uridyltransferase activity.

BackgroundClassic galactosemia (OMIM #230400) is an autosomal recessive metabolic disorder caused by a deficiency of the galactose-1-phosphate uridyltransferase (GALT, EC2.7.7.12) protein due to mutations in the GALT gene. The aim of this study was to provide a comprehensive and updated mutation spectrum of GALT in a Korean population.MethodsThirteen unrelated patients screened positive for galactosemia in a newborn screening program were included in this study. They showed a reduced GALT enzyme activity in red blood cells. Direct sequencing of the GALT gene and in silico analyses were done to evaluate the impact of novel variations upon GALT enzyme activity. We also reviewed previous reports for GALT mutations in Koreans.ResultsWe identified six novel likely pathogenic variations including three missense (p.Ala101Asp, p.Tyr165His, and p.Pro257Thr), one small deletion/insertion [c.826_827delinsAA (p.Ala276Asn)], one frameshift (p.Asn96Serfs*5), and one splicing (c.378-1G > C) likely pathogenic variations. The most frequent variation was the Duarte variant (c.940A > G, 35.3%), followed by c.507G > C (p.Gln169His, 9.6%), among 34 Korean patients. Other mutations were widely scattered. None of the eight common mutations used for targeted mutation analysis in Western countries including p.Gln188Arg, p.Ser135Leu, p.Lys285Asn, p.Leu195Pro, p.Tyr209Cys, p.Phe171Ser, c.253-2A > G, and a 5 kb deletion, had been found in Koreans until this study.ConclusionsConsidering the mutation spectrum in Koreans, direct sequence analysis of entire GALT exons is recommended for accurate diagnosis. The mutations responsible for GALT deficiency in the Korean population were clearly different from those of other populations.

Inhibition studies on Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase (GlmU)

Peptidoglycan is an essential component of the cell wall of bacteria, including Mycobacterium tuberculosis, that provides structural strength and rigidity to enable internal osmotic pressure to be withstood. The first committed step in the biosynthesis of peptidoglycan involves the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) from uridine triphosphate (UTP) and GlcNAc-1-phosphate. This reaction is catalysed by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme with two independent active sites that possess acetyltransferase and uridyltransferase activities. Herein, we report the first inhibition study targeted against the uridyltransferase activity of M. tuberculosis GlmU. A number of potential inhibitors were initially prepared leading to the discovery of active aminoquinazoline-based compounds. The most potent inhibitor in this series exhibited an IC50 of 74 μM against GlmU uridyltransferase activity and serves as a promising starting point for the discovery of more potent inhibitors.

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 Å. 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.