Substrate Phosphoenolpyruvate Research Articles

Rheostatic contributions to protein stability can obscure a position's functional role.

Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC.

There is growing interest in using isothermal titration calorimetry (ITC) to characterize enzyme kinetics by measuring the heat produced or absorbed by catalysis in real time. Since virtually all chemical reactions are associated with changes in enthalpy, ITC represents a robust and nearly universal experimental approach. Nevertheless, there are technical challenges that limit ITC's applicability. For instance, the full kinetic characterization of enzymes with two substrates (bi-substrate enzymes), which comprise the majority of known examples, requires a series of experiments to be performed as the concentrations of both substrates are varied. This is a time-consuming and expensive process using current ITC methods since many (>5) individual experiments must be performed independently to obtain a sufficient quantity of data. We have developed a new ITC method, which we term 2D-ITC, which maps the reaction velocity as a function of two substrate concentrations in a single, roughly 2 h long experiment. This method provides a level of detail that rivals or exceeds any existing enzyme assay, as a single experiment generates on the order of 7000 catalytic rate measurements. In a proof-of-principle application to rabbit muscle pyruvate kinase (rMPK), the method correctly identified the enzyme's random sequential mechanism and allosteric catalytic suppression by the amino acid phenylalanine (Phe). Unexpectedly, we found that while Phe reduces affinity for the substrate phosphoenolpyruvate, a known phenomenon, it also alleviates inhibition by the reaction product ATP, which had not been reported previously. Given the relative abundance of ATP in the cell, this opposing effect is expected to have a substantial impact on rMPK activity.

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase.

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PKTIM+Reg) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PKTIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PKTIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.

Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics.

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

Identification of the allosteric site for neutral amino acids in the maize C4 isozyme of phosphoenolpyruvate carboxylase: The critical role of Ser-100

The isozymes of photosynthetic phosphoenolpyruvate carboxylase from C4 plants (PEPC-C4) play a critical role in their atmospheric CO2 assimilation and productivity. They are allosterically activated by phosphorylated trioses or hexoses, such as d-glucose 6-phosphate, and inhibited by l-malate or l-aspartate. Additionally, PEPC-C4 isozymes from grasses are activated by glycine, serine, or alanine, but the allosteric site for these compounds remains unknown. Here, we report a new crystal structure of the isozyme from Zea mays (ZmPEPC-C4) with glycine bound at the monomer-monomer interfaces of the two dimers of the tetramer, making interactions with residues of both monomers. This binding site is close to, but different from, the one proposed to bind glucose 6-phosphate. Docking experiments indicated that d/l-serine or d/l-alanine could also bind to this site, which does not exist in the PEPC-C4 isozyme from the eudicot plant Flaveria, mainly because of a lysyl residue at the equivalent position of Ser-100 in ZmPEPC-C4 Accordingly, the ZmPEPC-C4 S100K mutant is not activated by glycine, serine, or alanine. Amino acid sequence alignments showed that PEPC-C4 isozymes from the monocot family Poaceae have either serine or glycine at this position, whereas those from Cyperaceae and eudicot families have lysine. The size and charge of the residue equivalent to Ser-100 are not only crucial for the activation of PEPC-C4 isozymes by neutral amino acids but also affect their affinity for the substrate phosphoenolpyruvate and their allosteric regulation by glucose 6-phosphate and malate, accounting for the reported kinetic differences between PEPC-C4 isozymes from monocot and eudicot plants.

Expression, purification, and characterization of pyruvate kinase from Mycobacterium tuberculosis: A key allosteric regulatory enzyme.

The current research aims to isolate pyruvate kinase (Pyk) gene from Mycobacterium tuberculosis and expression of the gene (Rv1617) to obtain a purified enzyme. The enzyme activity and secondary structural features were assessed through biochemical assays and circular dichroism (CD) spectroscopy, respectively. The Pyk-encoding gene from the complete genome of M. tuberculosis was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). The enzyme was purified by nickel-nitrilotriacetic acid affinity chromatography and enzyme activity was determined by a lactose dehydrogenase-coupled assay system. Further, far ultraviolet CD spectra of the enzyme and the substrate bound enzyme were analyzed using a Jasco J712 spectrophotometer. A single protein with an approximate molecular mass of 54 kDa was purified and a specific activity of 5.31 units/mg was determined from purified M. tuberculosis Pyk. The activity of the enzyme indicating a protein is defined by separate domain for each catalytic function. The secondary structure analysis of CD spectra of the recombinant Pyk has revealed a content of 17% α-helix, 34% β-sheet, and 49% turns in the enzyme. The growing evidence has impacted M. tuberculosis central carbon metabolism as a key determinant of the survival and pathogenicity in the host. The purified Pyk was observed to have increased enzyme activity in all steps of purification. Retention of Pyk activity indicates a possible catalytic role for the lower part of the glycolytic pathway. The overall results of the spectra obtained from the CD suggest that the substrate phosphoenolpyruvate and adenosine diphosphate binding to the enzyme can cause conformational changes resulting in the exposure or shielding of residues susceptible to modification.

Allosteric Regulation of the Photosynthetic C4 Isoenzyme of Phosphoenolpyruvate Carboxylase: A Comparative Study Between Enzymes from Monocot and Dicot Plants

In the present study, we have investigated the complex allosteric regulation of the non-phosphorylated forms of the photosynthetic phosphoenolpyruvate carboxylase isoenzymes (PEPC-C4) from amaranth (AhPEPC-C4) and maize (ZmPEPC-C4) leaves. Previous studies showed that glycine (Gly) only activates PEPC-C4 from monocot plants, as maize, but not from dicot plants, as amaranth. Our initial velocity data confirm this, in spite that AhPEPC-C4 binds Gly with much higher affinity than ZmPEPC-C4. In AhPEPC-C4, the lack of Gly activation is overcome mainly by its higher affinity for the substrate phosphoenolpyruvate and its lower affinity for the inhibitor malate compared with ZmPEPC-C4. We have also explored the structural determinants of the differences in Gly activation by performing multiple alignments between the known monocot and dicot PEPC-C4 sequences and by modeling, in both the AhPEPC-C4 and ZmPEPC-C4 isoenzymes, the three-dimensional structure of the loop proposed as the Gly binding site, which was not observed in the crystal structure of the maize enzyme due to its high flexibility. The models suggest that conserved lysyl and aspartyl residues are important for binding to the activator molecule, and that a nearby non-conserved residue may be responsible for differences between the amaranth and maize enzymes in the loop conformation, which would account for the poorer affinity for Gly of the maize enzyme as well as for its higher degree of activation.

Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells.

The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between non-transformed cells and tumor cells.

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I.

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here, we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate (PEP) and the inhibitor α-ketoglutarate (αKG), on the structure and dynamics of EI using NMR, small-angle X-ray scattering (SAXS) and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Comparison of Plant-Type Phospho enol pyruvate Carboxylases from Rice: Identification of Two Plant-Specific Regulatory Regions of the Allosteric Enzyme

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae and vascular plants, and it undergoes allosteric regulation by various metabolic effectors. Rice (Oryza sativa) has five plant-type PEPCs, four cytosolic and one chloroplastic. We investigated their kinetic properties using recombinant proteins and found that, like most plant-type PEPCs, rice cytosolic isozymes were activated by glucose 6-phosphate and by alkaline pH. In contrast, no such activation was observed for the chloroplastic isozyme, Osppc4. In addition, Osppc4 showed low affinity for the substrate phosphoenolpyruvate (PEP) and very low sensitivities to allosteric inhibitors aspartate and glutamate. By comparing the isozyme amino acid sequences and three-dimensional structures simulated on the basis of the reported crystal structures, we identified two regions where Osppc4 has unique features that can be expected to affect its kinetic properties. One is the N-terminal extension; replacement of the extension of Osppc2a (cytosolic) with that from Osppc4 reduced the aspartate and glutamate sensitivities to about one-tenth of the wild-type values but left the PEP affinity unaffected. The other is the N-terminal loop, in which a conserved lysine at the N-terminal end is replaced with a glutamate-alanine pair in Osppc4. Replacement of the lysine of Osppc2a with glutamate-alanine lowered the PEP affinity to a quarter of the wild-type level (down to the Osppc4 level), without affecting inhibitor sensitivity. Both the N-terminal extension and the N-terminal loop are specific to plant-type PEPCs, suggesting that plant-type isozymes acquired these regions so that their activity could be regulated properly at the sites where they function.

Characterization of a new type of glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase from Isoptericola variabilis

The 5-enolpyruvylshikimate-3-phosphate synthases (EPSPSs) encoded by aroA genes are traditionally divided into two classes, and only the class II EPSPS is naturally insensitive to herbicide glyphosate. Here, a novel EPSPS from Isoptericola variabilis, designated as I. variabilis EPSPS, was characterized. Its aroA gene contained 1374bp with 77% of GC contents, encoding a peptide of 458 amino acids. The EPSPS carried a highly conserved region involved in substrate bindings in EPSPSs and displayed a high sequence and structure similarity to class I EPSPS. However, this EPSPS had high naturally glyphosate-tolerant capability and enzymatic kinetics constants similar to that of class II EPSPSs. Escherichia coli AB2829 harboring the aroA gene grew well in medium containing 150mM glyphosate. The purified EPSPS had the highest enzymatic activity at pH 8.0 and 30°C, and retained its activity at pH 6–10 and 20–60°C. This enzyme had a Km value of 28μM for substrate phosphoenolpyruvate and 111μM for shikimate-3-phosphate, respectively, with a rather high Ki value for glyphosate. Cations increased substrate affinity but slightly reduced glyphosate tolerance. The I. variabilis EPSPS may be used for generating transgenic glyphosate-tolerant plants and dissecting mechanisms underlying the glyphosate tolerance.

Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK–TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.

Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum

Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg.

Distinguishing the Chemical Moiety of Phosphoenolpyruvate That Contributes to Allostery in Muscle Pyruvate Kinase

A series of substrate analogues has been used to determine which chemical moieties of the substrate phosphoenolpyruvate (PEP) contribute to the allosteric inhibition of rabbit muscle pyruvate kinase by phenylalanine. Replacing the carboxyl group of the substrate with a methyl alcohol or removing the phosphate altogether greatly reduces substrate affinity. However, removal of the carboxyl group is the only modification tested that removes the ability to allosterically reduce the level of Phe binding. From this, it can be concluded that the carboxyl group of PEP is responsible for energetic coupling with Phe binding in the allosteric sites.

M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth

Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.

Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA( P.fluorescens ) indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K ( i ) for glyphosate compared to those of the AroA( E.coli ), while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA ( P.fluorescens ) gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.

A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.

Functional Consequence of Covalent Reaction of Phosphoenolpyruvate with UDP-N-acetylglucosamine 1-Carboxyvinyltransferase (MurA)

The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.