Conserved cytoplasmic motifs that distinguish sub-groups of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family

WecA, MraY and WbcO are conserved members of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family involved in the assembly of bacterial cell walls, and catalyze reactions involving a membrane-associated polyprenol phosphate acceptor substrate and a cytoplasmically located UDP-D-amino sugar donor. MraY, WbcO and WecA purportedly utilize different UDP-sugars, although the molecular basis of this specificity is largely unknown. However, domain variations involved in specificity are predicted to occur on the cytoplasmic side of the membrane, adjacent to conserved domains involved in the mechanistic activity, and with access to the cytoplasmically located sugar nucleotides. Conserved C-terminal domains have been identified that satisfy these criteria. Topological analyses indicate that they form the highly basic, fifth cytoplasmic loop between transmembrane regions IX and X. Four diverse loops are apparent, for MraY, WecA, WbcO and RgpG, that uniquely characterize these sub-groups of the transferase family, and a correlation is evident with the known or implied UDP-sugar specificity.     

Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases

Protein N-glycosylation in eukaryotes and peptidoglycan biosynthesis in bacteria are both initiated by the transfer of a D-N-acetylhexosamine 1-phosphate to a membrane-bound polyprenol phosphate. These reactions are catalyzed by a family of transmembrane proteins known as the UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-phosphate transferases. The sole eukaryotic member of this family, the d-N-acetylglucosamine 1-phosphate transferase (GPT), is specific for UDP-GlcNAc as the donor substrate and uses dolichol phosphate as the membrane-bound acceptor. The bacterial translocases, MraY, WecA, and WbpL, utilize undecaprenol phosphate as the acceptor substrate, but differ in their specificity for the UDP-sugar donor substrate. The structural basis of this sugar nucleotide specificity is uncertain. However, potential carbohydrate recognition (CR) domains have been identified within the C-terminal cytoplasmic loops of MraY, WecA, and WbpL that are highly conserved in family members with the same UDP-N-acetylhexosamine specificity. This review focuses on the catalytic mechanism and substrate specificity of these bacterial UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-P transferases and may provide insights for the development of selective inhibitors of cell wall biosynthesis.     

Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase

Bacterial peptidoglycan synthesis is a well-characterized system for targeting new antimicrobial drugs. Formation of the peptidoglycan precursors Lipid I and Lipid II is catalyzed by the gene products of mraY and murG, which are involved in the first and second steps of the lipid cycle reactions, respectively. Here we describe the development of an assay specific for identifying inhibitors of MraY or MurG, based on the detection of radiolabeled [(14)C]GlcNAc incorporated into Lipid II. Assay specificity is achieved with the biotin tagging of the Lipid I precursor UDP-MurNAc-pentapeptide. This allows for the separation and identification of lipid products produced by the enzymatic activity of the MraY and MurG proteins, and thus identification of specific inhibitors.     

The biosynthesis of peptidoglycan lipid-linked intermediates

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.     

Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.     

Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis

The MraY translocase catalyzes the first membrane step of bacterial cell wall peptidoglycan synthesis (i.e. the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid), a reversible reaction yielding undecaprenylpyrophosphoryl-N-acetylmuramoyl-pentapeptide (lipid intermediate I). This essential integral membrane protein, which is considered as a very promising target for the search of new antibacterial compounds, has thus far been clearly underexploited due to its intrinsic refractory nature to overexpression and purification. We here report conditions for the high level overproduction and for the first time the purification to homogeneity of milligram quantities of MraY protein. The kinetic parameters and effects of pH, salts, cations, and detergents on enzyme activity are described, taking the Bacillus subtilis MraY translocase as a model.     

Development of a microplate-based scintillation proximity assay for MraY using a modified substrate

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

The Methanococcus jannaschii dCTP deaminase is a bifunctional deaminase and diphosphatase

Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg(2+). The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions.     

A high-throughput, homogeneous, fluorescence resonance energy transfer-based assay for phospho-N-acetylmuramoyl-pentapeptide translocase (MraY)

Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product-derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer-based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.     

De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase

DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3'OH end of the primer along the template. On the other hand terminal deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3'OH end of a DNA strand, without the need of a template. Pol lambda and pol micro are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol micro and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol micro and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.     

Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).     

Structural insights into catalysis and inhibition of O-acetylserine sulfhydrylase from Mycobacterium tuberculosis. Crystal structures of the enzyme alpha-aminoacrylate intermediate and an enzyme-inhibitor complex

Cysteine biosynthetic genes are up-regulated in the persistent phase of Mycobacterium tuberculosis, and the corresponding enzymes are therefore of interest as potential targets for novel antibacterial agents. cysK1 is one of these genes and has been annotated as coding for an O-acetylserine sulfhydrylase. Recombinant CysK1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine to cysteine. The crystal structure of the enzyme was determined to 1.8A resolution. CysK1 belongs to the family of fold type II PLP enzymes and is similar in structure to other O-acetylserine sulfhydrylases. We were able to trap the alpha-aminoacrylate reaction intermediate and determine its structure by cryocrystallography. Formation of the aminoacrylate complex is accompanied by a domain rotation resulting in active site closure. The aminoacrylate moiety is bound in the active site via the covalent linkage to the PLP cofactor and by hydrogen bonds of its carboxyl group to several enzyme residues. The catalytic lysine residue is positioned such that it can protonate the Calpha-carbon atom of the aminoacrylate only from the si-face, resulting in the formation of L-cysteine. CysK1 is competitively inhibited by a four-residue peptide derived from the C-terminal of serine acetyl transferase. The crystallographic analysis reveals that the peptide binds to the enzyme active site, suggesting that CysK1 forms an bi-enzyme complex with serine acetyl transferase, in a similar manner to other bacterial and plant O-acetylserine sulfhydrylases. The structure of the enzyme-peptide complex provides a framework for the design of strong binding inhibitors.     

Functional Diversity of Xyloglucan-Related Proteins and its Implications in the Cell Wall Dynamics in Plants

Abstract: The plant cell wall is a dynamic apparatus responsible for both morphogenesis and responsiveness to environmental conditions. In the cell wall of most seed plants, cellulose microfibrils are cross-linked by xyloglucans to form a cellulose/xyloglucan framework, which functions as the mechanical underpinning of the cell wall. Endoxyloglucan transferases are a class of enzymes that play a central role in construction and modification of the plant cell wall. These enzymes are encoded by a large multi-gene family termed xyloglucan-related proteins (XRPs). More than 24 members of the XRP family have so far been identified in Arabidopsis thaliana. Each member of this family functions as either a hydrolase or a transferase acting on xyloglucans. The primary structures of proteins and gene-expression profiles have strongly suggested their potentially divergent roles in plant morphogenesis: different members of this family are expressed in different types of tissues at distinct developmental stages and respond differentially to individual hormones as well as environmental stimuli. These facts imply that each member of this gene family is individually committed to a specific process that proceeds in a specific tissue at a specific stage of development. Probably the generation and maintenance of the cell walls in a whole organ, and thus in the whole plant, is achieved by the ensemble of individual members of the XRP family.     

An end-healing enzyme from Clostridium thermocellum with 5' kinase, 2',3' phosphatase, and adenylyltransferase activities

We identify and characterize an end-healing enzyme, CthPnkp, from Clostridium thermocellum that catalyzes the phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and the dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp also catalyzes an autoadenylylation reaction via a polynucleotide ligase-type mechanism. These characteristics are consistent with a role in end-healing during RNA or DNA repair. CthPnkp is a homodimer of an 870-amino-acid polypeptide composed of three catalytic domains: an N-terminal module that resembles the polynucleotide kinase domain of bacteriophage T4 Pnkp, a central metal-dependent phosphoesterase module, and a C-terminal module that resembles the nucleotidyl transferase domain of polynucleotide ligases. The distinctive feature of CthPnkp vis-à-vis known RNA repair enzymes is that its 3' end modification component belongs to the calcineurin-type phosphatase superfamily. It contains putative counterparts of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. As with lambda phosphatase, the 2',3' cAMP phosphatase activity of CthPnkp is specifically dependent on nickel or manganese. We identify homologs of CthPnkp in other bacterial proteomes.     

S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1

Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer’s disease and the posttranslational activation of interleukin 1β (IL-1β). Investigation of the biological role of GSTO1-1 variants has been hampered by the lack of a specific assay for GSTO1-1 activity in tissue samples that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues.     

Flavocytochrome b2: reactivity of its flavin with molecular oxygen

Flavocytochrome b2, a flavohemoprotein, catalyzes the oxidation of lactate at the expense of the physiological acceptor cytochrome c in the yeast mitochondrial intermembrane space. The mechanism of electron transfer from the substrate to monoelectronic acceptors via FMN and heme b2 has been intensively studied over the years. Each prosthetic group is bound to a separate domain, N-terminal for the heme, C-terminal for the flavin. Each domain belongs to a distinct evolutionary family. In particular, the flavodehydrogenase domain is homologous to a number of well-characterized l-2-hydroxy acid-oxidizing enzymes. Among these, some are oxidases for which the oxidative half-reaction produces hydrogen peroxide at the expense of oxygen. For bacterial mandelate dehydrogenase and flavocytochrome b2, in contrast, the oxidative half-reaction requires monoelectronic acceptors. Several crystal structures indicate an identical fold and a highly conserved active site among family members. All these enzymes form anionic semiquinones and bind sulfite, properties generally associated with oxidases, whereas electron transferases are expected to form neutral semiquinones and to yield superoxide anion. Thus, flavocytochrome b2 is a highly unusual dehydrogenase-electron transferase, and one may wonder how its flavin reacts with oxygen. In this work, we show that the separately engineered flavodehydrogenase domain produces superoxide anion in its slow reaction with oxygen. This reaction apparently also takes place in the holoenzyme when oxygen is the sole electron acceptor, because the heme domain autoxidation is also slow; this is not unexpected, in view of the heme domain mobility relative to the tetrameric flavodehydrogenase core (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863). Nevertheless, this reaction is so slow that it cannot compete with the normal electron flow in the presence of monoelectronic acceptors, such as ferricyanide and cytochrome c. An inspection of the available structures of family members does not provide a rationale for the difference between the oxidases and the electron transferases.     

Bacterial transferase MraY inhibitors: Synthesis and biological evaluation

New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interesting inhibitory activity for both amino compounds.     

Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates

Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.     

Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX4DX6G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 10(4) and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.