Phosphate Transfer Reactions Research Articles

On the Computational Power of Phosphate Transfer Reaction Networks

Phosphate transfer reactions (Principles of biochemistry, Prentice Hall, Upper Saddle River, 1996) involve the transfer of a phosphate group from a donor molecule to an accepter, which is ubiquitous in biochemistry. Besides natural systems, some synthetic molecular systems such as seesaw gates are also equivalent to (subsets of) phosphate transfer reaction networks. In this paper, we study the computational power of phosphate transfer reaction networks (PTRNs). PTRNs are chemical reaction networks (CRNs) with only phosphate transfer reactions. Previously, it is known (Nat Comput 13:517–534, 2014) that a function can be deterministically computed by a CRN if and only if it is semilinear. However, the computational power of programmable phosphate transfer networks is unknown. In this paper, we present a formal model to describe PTRNs and study the computational power of these networks. We prove that when each molecule can only carry one phosphate group, the output must be the total initial count in a subset \(S_1\) minus the total initial count of another subset \(S_2\). On the other hand, when every molecule can carry up to three phosphate groups, or two phosphate groups with different functions, PTRNs can “simulate” arbitrary CRNs. Finally, when each molecule can carry up to two functionally identical phosphate groups (or, equivalently, two phosphate groups which must be added/removed in a sequential manner), we prove that the computational power is strictly stronger than PTRNs with at most one phosphate group per molecule.

Conformational and Reaction Dynamic Coupling in Histidine Kinases: Insights from Hybrid QM/MM Simulations.

Histidine kinases (HK) of bacterial two-component systems represent a hallmark of allosterism in proteins, being able to detect a signal through the sensor domain and transmit this information through the protein matrix to the kinase domain which, once active, autophosphorylates a specific histidine residue. Inactive-to-active transition results in a large conformational change that moves the kinase on top of the histidine. In the present work, we use several molecular simulation techniques (Molecular Dynamics, Hybrid QM/MM, and constant pH molecular dynamics) to study the activation and autophosphorylation reactions in L. plantarum WalK, a cis-acting HK. In agreement with previous results, we show that the chemical step requires tight coupling with the conformational step in order to maintain the histidine phosphoacceptor in the correct tautomeric state, with a reactive δ-nitrogen. During the conformational transition, the kinase domain is never released and walks along the HK helix axis, breaking and forming several conserved residue-based contacts. The phosphate transfer reaction is concerted in the transition state region and is catalyzed through the stabilization of the negative developing charge of transferring phosphate along the reaction.

Phosphorylation Mechanism of N-Acetyl-l-glutamate Kinase, a QM/MM Study.

In microorganisms and plants, N-acetyl-l-glutamate kinase (NAGK) catalyzes the second step in l-arginine synthesis, the phosphorylation of N-Acetyl-l-glutamate (NAG) to give N-acetyl-l-glutamate-5-phosphate. NAGK is only present in microorganisms and plants but absent in mammals, which makes it an attractive target for antimicrobial or biocidal development. Understanding the substrate binding mode and reaction mechanism of NAGK is crucial for targeting the kinase to develop potential therapies. Here, the substrate binding mode was studied by comparing the conformational change of NAGK in the presence and in the absence of the NAG substrate based on molecular dynamics simulations. We revealed that with substrate binding, the catalytic site of the kinase involving three loops in NAGK exhibits a closed conformation, which is predominantly controlled by an interaction between Arg98 and the α-COO- of NAG. Lys41 is found to guide phosphate transfer through the interactions with the β-,γ-, and γ-phosphate oxygen atoms of adenosine 5'-triphosphate surrounded by two highly conserved glycine residues (Gly44 and Gly76), while Arg98 helps to position the NAG substrate in the catalytic site, which facilitates the phosphate transfer. Furthermore, we elucidated phosphate-transfer reaction mechanism using hybrid density functional theory-based quantum mechanics/molecular mechanics calculations (B97D/AMBER99) and found that the catalysis follows a dissociative mechanism.

Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in Saccharomyces cerevisiae.

Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.

Frank Henry Westheimer. 15 January 1912—14 April 2007

Frank H. Westheimer integrated physical and organic chemistry to become an important contributor to the field that came to be called ‘physical organic chemistry’. He then went on to apply physical organic chemistry to the study of biological transformations. He identified fundamental classes of chemical reactions, studied in detail how they occurred in a relevant non-biological format and then investigated corresponding enzymatic reactions. These included oxidation–reduction, decarboxylation and phosphate transfer reactions. In the process, he made fundamental observations in the field of bio-organic chemistry he had helped to create. This line of research led to conceptual advances relevant to the ability of enzymes to distinguish enantiotopic and diastereotopic centres in their substrates, the perturbation of p K a values in enzymatic active sites as a source of catalytic power, and the broad range of transition state structures available for reactions at electrophilic phosphorus. These studies prompted similar work across many classes of enzymatic reactions. These experiments also benefitted from Westheimer's guidance in the application of kinetic isotope effects to probe reaction transition states, his invention of molecular mechanics to understand molecular dynamics, his widely cast studies on how organic reactivity depends on the molecular environment and his demonstration that photochemistry could be used to probe structure in complex biological systems.

QM/MM Study of the Dephosphorylation Mechanism of Adenosine 5′‐(β,γ‐Imido)triphosphate Catalyzed by Sulfolobus Tokodaii Hexokinase

AbstractAdenosine triphosphate (ATP) bound hexokinase crystal structure is important for understanding the functions of hexokinase at the atomic level, but it is hard to obtain such complex because of the easy hydrolysis of ATP. Adenosine 5′‐(β,γ‐imido)triphosphate (AMP‐PNP) is a commonly used non‐hydrolysable ATP analogue for co‐crystallization with many proteins, but the AMP‐PNP function towards hexokinase is still unknown in the literature. In the present paper, we would like to report the dephosphorylation mechanisms of AMP‐PNP catalyzed by the wild‐type Sulfolobus tokodaii hexokinase (StHK) and T116A mutant using quantum mechanical/molecular mechanical (QM/MM) method. The catalytic reactions proceed via a phosphate transfer, followed by a proton transfer in these models. Both StHK and T116A mutant catalyzed reactions are endothermic. The phosphate transfer reaction is the rate‐determining step, and their energy barriers are 15.95 and 22.71 kcal⋅mol−1 for StHK and T116A mutant, respectively. Comparison of the ATP dephosphorylation shows that the catalytic processes by the wild‐type and T116A mutated StHK are passivated. The passivation has been attributed to the linking –NH group of AMP‐PNP. The results are meaningful to a better understand of hexokinase and the functions of AMP‐PNP in cocrystallization.

Phosphate Transfer in Activated Protein Complexes Reveals Interaction Sites.

For many proteins, phosphorylation regulates their interaction with other biomolecules. Herein, we describe an unexpected phenomenon whereby phosphate groups are transferred non-enzymatically from one interaction partner to the other within a binding interface upon activation in the gas phase. Providing that a high affinity exists between the donor and acceptor sites, this phosphate transfer is very efficient and the phosphate groups only ligate to sites in proximity to the binding region. Consequently, such phosphate-transfer reactions may define with high precision the binding site between a phosphoprotein and its binding partner, as well as reveal that the binding site in this system is retained in the phase transfer from solution to the gas phase.

Has Inositol Played Any Role in the Origin of Life?

Phosphorus, as phosphate, plays a paramount role in biology. Since phosphate transfer reactions are an integral part of contemporary life, phosphate may have been incorporated into the initial molecules at the very beginning. To facilitate the studies into early phosphate utilization, we should look retrospectively to phosphate-rich molecules present in today’s cells. Overlooked by origin of life studies until now, inositol and the inositol phosphates, of which some species possess more phosphate groups that carbon atoms, represent ideal molecules to consider in this context. The current sophisticated association of inositol with phosphate, and the roles that some inositol phosphates play in regulating cellular phosphate homeostasis, intriguingly suggest that inositol might have played some role in the prebiotic process of phosphate exploitation. Inositol can be synthesized abiotically and, unlike glucose or ribose, is chemically stable. This stability makes inositol the ideal candidate for the earliest organophosphate molecules, as primitive inositol phosphates. I also present arguments suggesting roles for some inositol phosphates in early chemical evolution events. Finally, the possible prebiotic synthesis of inositol pyrophosphates could have generated high-energy molecules to be utilized in primitive trans-phosphorylating processes.

Supramolecular Polymer/Surfactant Complexes as Catalysts for Phosphate Transfer Reactions

Designing artificial enzymes with tailored molecular interactions between the substrate and active site is of major intellectual and practical significance. We report the improved catalytic efficiency of a supramolecular polymer/surfactant complex comprised of PAIM–, a poly(acrylic acid) derivative with imidazole groups attached to the polymer by amide bonds, and the cationic surfactant cetyltrimethylammonium bromide (CTAB). Supramolecular complex formation, at concentrations below the respective CMC values, provides convenient hydrophobic pockets for the reactants close to the multiple catalytic centers, where imidazole and carboxylate groups act as nucleophiles for the degradation of a model phosphate triester, delivering the highly efficient performance of the supramolecular catalysts. Catalytic effects are on the order of thousands for nucleophilic catalysis and are higher by 2 orders of magnitude for the supramolecular polymer/surfactant complex at pH 9. The reported supramolecular catalytic complexe...

Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

Gas-Phase Rearrangement in Lysine Phosphorylated Peptides During Electron-Transfer Dissociation Tandem Mass Spectrometry.

Tandem mass spectrometry (MS/MS) strategies coupled with collision-induced dissociation (CID) or radical-driven fragmentation techniques such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) have been successfully used for comprehensive phosphoproteome analysis. However, the unambiguous characterization of the phosphorylation site remains a significant challenge due to phosphate-related neutral losses and gas-phase rearrangements, which have been observed during CID. In particular, for the analysis of labile N-phosphorylated peptides, ECD and ETD are emerging as a complementary method. In contrast to CID, the phosphorylation site of histidine, arginine, and lysine phosphorylated peptides can be characterized by ETD. Here, we present a study on the application of ETD for analysis of phospholysine (pLys) peptides. We show that, depending on the charge state of the precursor ion as well as the presence of basic amino acid side chains, phosphate transfer reactions during the ETD process can be observed leading to ambiguous fragment ion spectra. Basically, pLys is stable under ETD conditions allowing an unambiguous assignment of the site of phosphorylation, but some factors/parameters have to be considered to avoid gas-phase rearrangement which would lead to false positive results in phosphoproteomic studies.

Fundamentals of phosphate transfer.

Historically, the chemistry of phosphate transfer-a class of reactions fundamental to the chemistry of Life-has been discussed almost exclusively in terms of the nucleophile and the leaving group. Reactivity always depends significantly on both factors; but recent results for reactions of phosphate triesters have shown that it can also depend strongly on the nature of the nonleaving or "spectator" groups. The extreme stabilities of fully ionised mono- and dialkyl phosphate esters can be seen as extensions of the same effect, with one or two triester OR groups replaced by O(-). Our chosen lead reaction is hydrolysis-phosphate transfer to water: because water is the medium in which biological chemistry takes place; because the half-life of a system in water is an accepted basic index of stability; and because the typical mechanisms of hydrolysis, with solvent H2O providing specific molecules to act as nucleophiles and as general acids or bases, are models for reactions involving better nucleophiles and stronger general species catalysts. Not least those available in enzyme active sites. Alkyl monoester dianions compete with alkyl diester monoanions for the slowest estimated rates of spontaneous hydrolysis. High stability at physiological pH is a vital factor in the biological roles of organic phosphates, but a significant limitation for experimental investigations. Almost all kinetic measurements of phosphate transfer reactions involving mono- and diesters have been followed by UV-visible spectroscopy using activated systems, conveniently compounds with good leaving groups. (A "good leaving group" OR* is electron-withdrawing, and can be displaced to generate an anion R*O(-) in water near pH 7.) Reactivities at normal temperatures of P-O-alkyl derivatives-better models for typical biological substrates-have typically had to be estimated: by extended extrapolation from linear free energy relationships, or from rate measurements at high temperatures. Calculation is free from these limitations, able to handle very slow reactions as readily as very fast ones, and capable of predicting rate constants with levels of accuracy acceptable to the experimentalist. We present an updated overview of phosphate transfer, with particular reference to the mechanisms of the reactions of alkyl derivatives and triesters. The intention is to present a holistic (not comprehensive!) overview of the reactivity of typical phosphate esters, in terms familiar to the working chemist, at a level sufficient to support informed predictions of reactivity for structures of interest.

Activating Water: Important Effects of Non‐leaving Groups on the Hydrolysis of Phosphate Triesters

The high rate of spontaneous hydrolysis of tris-2-pyridyl phosphate (TPP) is explained by the activating effects of the non-leaving ("spectator") groups on P-OAr cleavage, and not by intramolecular catalysis. Previous work on phosphate-transfer reactions has concentrated on the contributions to reactivity of the nucleophile and the leaving group, but our results make clear that the effects of the non-leaving groups on phosphorus can be equally significant. Rate measurements for three series of phosphate triesters showed that sensitivities to the non-leaving groups are substantial for spontaneous hydrolysis reactions, although significantly smaller for reactions with good nucleophiles. There are clear differences between triaryl and dialkyl aryl triesters in sensitivities to leaving and non-leaving groups with the more reactive triaryl systems showing lower values for both β(LG) and β(NLG). Intramolecular catalysis of the hydrolysis of TPP by the neighbouring pyridine nitrogens is insignificant, primarily because of their low basicity.

Phosphoenolpyruvate and Mg2+ Binding to Pyruvate Kinase Monitored by Infrared Spectroscopy

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg2+ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg2+ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-13C2-labeled PEP. We obtained the following assignments: 1589 cm−1 (antisymmetric carboxylate stretching vibration); 1415 cm−1 (symmetric carboxylate stretching vibration); 1214 cm−1 (C-O stretching vibration); 1124 and 1110 cm−1 (asymmetric PO32- stretching vibrations); and 967 cm−1 (symmetric PO32- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm−1. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na+ exhibited the same band positions as in the presence of K+, indicating very similar interaction strengths between ligand and protein in both cases.

Combining Medium Effects and Cofactor Catalysis: Metal‐Coordinated Synzymes Accelerate Phosphate Transfer by 108

The systematic exploration of the modification of polyethylene imine with guanidinium and octyl groups has led to the identification of a catalyst, CD6, which accelerates the phosphate transfer reaction of HPNP (2-hydroxypropyl-4-nitrophenyl phosphate) in the presence of divalent metals such as Zn(2+), Co(2+), Mg(2+) or Ni(2+). CD6 exhibits saturation kinetics that are described by Michaelis-Menten parameters K(m) ranging from 2.5-8 mM and k(cat) ranging from 0.0014-0.09 s(-1). For Zn(II)-CD6 this corresponds to an overall acceleration k(cat)/k(uncat) of 3.8x10(5) and a catalytic proficiency (k(cat)/K(m))/k(uncat) of 1.5x10(8). Catalysis by Zn(II)-CD6 is specifically inhibited by inorganic phosphate, allowing turnover regulation by product inhibition. This effect stands in contrast to Zn(II)-catalysed transesterification of HPNP in water or by the synzymes Co(II)-CD6 and Ni(II)-CD6, with which no such interference by product is observed. These characteristics render synzyme Zn(II)-CD6 an efficient enzyme model that reflects enzyme-like properties in a wide range of features.

Quantum mechanics/molecular mechanics investigation of the mechanism of phosphate transfer in human uridine-cytidine kinase 2

The mechanisms of enzyme-catalyzed phosphate transfer and hydrolysis reactions are of great interest due to their importance and abundance in biochemistry. The reaction may proceed in a stepwise fashion, with either a pentavalent phosphorane or a metaphosphate anion intermediate, or by a concerted SN2 mechanism. Despite much theoretical work focused on a few key enzymes, a consensus for the mechanism has not been reached, and examples of all three possibilities have been demonstrated. We have investigated the mechanism of human uridine-cytidine kinase 2 (UCK2, EC 2.7.1.48), which catalyzes the transfer of a phosphate group from ATP to the ribose 5'-hydroxyl of cytidine and uridine. UCK2 is normally expressed in human placenta, but is overexpressed in certain cancer cells, where it is responsible for activating a class of antitumor prodrugs. The UCK2 mechanism was investigated by generating a 2D potential energy surface as a function of the P-O bonds forming and breaking, with energies calculated using a quantum mechanics/molecular mechanics potential (B3LYP/6-31G(d):AMBER). The mechanism of phosphate transfer is shown to be concerted, and is accompanied by concerted proton transfer from the 5'-hydroxyl to a conserved active site aspartic acid that serves as a catalytic base. The calculated barrier for this reaction is 15.1 kcal/mol, in relatively good agreement with the experimental barrier of 17.5 kcal/mol. The interactions of the enzyme active site with the reactant, transition state, and product are examined for their implications on the design of anticancer prodrugs or positron emission tomography (PET) reporter probes for this enzyme.

Description of pentacoordinated phosphorus under an external electric field: which basis sets and semi-empirical methods are needed?

Phosphate transfer reactions are ubiquitous in nature and play fundamental roles in ATP hydrolysis and protein phosphorylation processes. The mechanisms of these reactions involve a pentacoordinated phosphorus atom that can be an intermediate or a transition state. These structures are very sensitive to both internal and external electrostatic effects and their description with quantum mechanical methods is challenging. We have investigated the variations of geometry an energetics under an external electric field for two different molecules and their transition states of formation. The DFT method, with the mPW1PW91 functional employing several basis sets, and different semi-empirical methods have been tested. Compared to zero-field cases, one needs more extended basis sets to achieve the same precision. A good compromise for large systems is the 6-31+G(d). Many semi-empirical methods are unable to describe polarisation effects in pentacoordinated structures. The best methods to describe geometries are PM6 and AM1/d-PhoT and for energetics AM1/d-PhoT. Methods without d orbitals have poorer performances but the best among those is the AM1 parametrization of Arantes et al (Phys. Chem. Chem. Phys., 2006, 8, 347).

Structural dynamics of the Ca2+-ATPase studied by time-resolved infrared spectroscopy

This review discusses the contribution of time-resolved infrared spectroscopy to the understanding of the Ca2+pump in the sarcoplasmic reticulum membrane of skeletal muscle cells (SERCA1a). The focus is on interactions of the substrate ATP with the ATPase and on the bond parameters of the phosphoenzyme phosphate group. Functional groups throughout the ATP molecule are important for stabilising the closed conformation of the ATP–ATPase complex and for fast phosphorylation of the ATPase. Dissociation of the reaction product ADP after phosphorylation leads to a more open average conformation of the enzyme and does not trigger the transition from the first phosphoenzyme Ca2E1P to the second E2P. The P–O bond between phosphate and aspartyl moieties is weaker in Ca2E1P and E2P than in acetyl phosphate in aqueous solution, which explains the high reactivity of the phosphoenzymes. This ground state property of the phosphoenzymes prepares for a phosphate transfer reaction with dissociative character.

Structural dynamics of the Ca2+-ATPase studied by time-resolved infrared spectroscopy

This review discusses the contribution of time-resolved infrared spectroscopy to the understanding of the Ca 2+ pump in the sarcoplasmic reticulum membrane of skeletal muscle cells (SERCA1a). The focus is on interactions of the substrate ATP with the ATPase and on the bond parameters of the phosphoenzyme phosphate group. Functional groups throughout the ATP molecule are important for stabilising the closed conformation of the ATP-ATPase complex and for fast phosphorylation of the ATPase. Dissociation of the reaction product ADP after phosphorylation leads to a more open average conformation of the enzyme and does not trigger the transition from the first phosphoenzyme Ca2E1P to the second E2P. The P-O bond between phosphate and aspartyl moieties is weaker in Ca2E1P and E2P than in acetyl phosphate in aqueous solution, which explains the high reactivity of the phosphoenzymes. This ground state property of the phosphoenzymes prepares for a phosphate transfer reaction with dissociative character.

Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. 32P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo2-[4′-32P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.