Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.

The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.

[Bridge between Total Synthesis of Bioactive Natural Products and Development of Drug Leads].

Although natural products are rich sources for drug discovery, only a small percentage of natural products themselves have been approved for clinical use, thus it is necessary to modulate various properties, such as efficacy, toxicity, and metabolic stability. A question in natural product drug discovery is how to logically design natural product derivatives with desired biological properties. This review describes our recent studies regarding the medicinal chemistry of tunicamycin. Tunicamycin inhibits bacterial phospho-N-acetylmuramic acid (MurNAc)-pentapeptide translocase (MraY), which is an essential enzyme in bacteria and a good target for antibacterial drug discovery. The usefulness of tunicamycin as antibacterial agents is limited by off-target inhibition of human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). We positioned the total synthesis of tunicamycin as a starting point for the research and have accomplished the synthesis of tunicamycin V by using the Achmatowicz reaction, [3,3] sigmatropic rearrangement of allyl cyanate, and stereoselective glycosylation as key reactions. Next, the minimum structural requirements for tunicamycin V for MraY inhibition were established by systematic structure-activity relationship studies with truncated analogs of tunicamycin V. Our collaborative study elucidated a crystal structure of human GPT in complex with tunicamycin. This structural information was then exploited to rationally design an MraY-specific inhibitor of tunicamycin V in which the GlcNAc moiety was modified to a MurNAc amide. The analog was identified as a highly selective MraYAA inhibitor.

Using Steady-State Kinetics to Quantitate Substrate Selectivity and Specificity: A Case Study with Two Human Transaminases.

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the kcat/KM values for two alternative substrates (the 'discrimination index'). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases' observed selectivities were remarkably high. In particular, GOT1 reacted ~106-fold less efficiently when the side-chain carboxylate of the 'physiological' substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using kcat/KM values to derive a global measure of enzyme specificity.

Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.

GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2ß2γ2 hexamers in which the core catalytic α- and ß-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase.

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

Synthetic Sansanmycin Analogues as Potent Mycobacterium tuberculosis Translocase I Inhibitors.

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity.

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.

A Mechanistic Basis for Phosphoethanolamine Modification of the Cellulose Biofilm Matrix in Escherichia coli.

Biofilms are communities of self-enmeshed bacteria in a matrix of exopolysaccharides. The widely distributed human pathogen and commensal Escherichia coli produces a biofilm matrix composed of phosphoethanolamine (pEtN)-modified cellulose and amyloid protein fibers, termed curli. The addition of pEtN to the cellulose exopolysaccharide is accomplished by the action of the pEtN transferase, BcsG, and is essential for the overall integrity of the biofilm. Here, using the synthetic co-substrates p-nitrophenyl phosphoethanolamine and ß-d-cellopentaose, we demonstrate using an in vitro pEtN transferase assay that full activity of the pEtN transferase domain of BcsG from E. coli (EcBcsGΔN) requires Zn2+ binding, a catalytic nucleophile/acid-base arrangement (Ser278/Cys243/His396), disulfide bond formation, and other newly uncovered essential residues. We further confirm that EcBcsGΔN catalysis proceeds by a ping-pong bisubstrate-biproduct reaction mechanism and displays inefficient kinetic behavior (kcat/KM = 1.81 × 10-4 ± 2.81 × 10-5 M-1 s-1), which is typical of exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected by KM), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes, which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family, including the intrinsic and mobile colistin resistance factors.

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds.

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.

Crossing the lipid divide.

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.

A promiscuous archaeal cardiolipin synthase enables construction of diverse natural and unnatural phospholipids.

Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain-based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif-containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is nonselective to the stereochemistry of the glycerol backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase that could be of interest for bio-catalytic purposes.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Structures of Bacterial MraY and Human GPT Provide Insights into Rational Antibiotic Design.

The widespread emergence of antibiotic resistance in pathogens necessitates the development of antibacterial agents inhibiting underexplored targets in bacterial metabolism. One such target is phospho-MurNAc-pentapeptide translocase (MraY), an essential integral membrane enzyme that catalyzes the first committed step of peptidoglycan biosynthesis. MraY has long been considered a promising candidate for antibiotic development in part because it is the target of five classes of naturally occurring nucleoside inhibitors with potent in vivo and in vitro antibacterial activity. Although these inhibitors each have a nucleoside moiety, they vary dramatically in their core structures, and they have different activity properties. Until recently, the structural basis of MraY inhibition was poorly understood. Several recent structures of MraY and its human paralog, GlcNAc-1-P-transferase, have provided insights into MraY inhibition that are consistent with known inhibitor activity data and can inform rational drug design for this important antibiotic target.

Conformational flexibility of coenzyme A and its impact on the post-translational modification of acyl carrier proteins by 4'-phosphopantetheinyl transferases.

One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4'-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4'-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases.

Diacylglycerol kinase δ and sphingomyelin synthase-related protein functionally interact via their sterile α motif domains.

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

Combined in vitro and in silico analyses of missense mutations in GNPTAB provide new insights into the molecular bases of mucolipidosis II and III alpha/beta.

Mucolipidosis (ML) II and III alpha/beta are inherited lysosomal storage disorders caused by mutations in GNPTAB encoding the α/ß-precursor of GlcNAc-1-phosphotransferase. This enzyme catalyzes the initial step in the modification of more than 70 lysosomal enzymes with mannose 6-phosphate residues to ensure their intracellular targeting to lysosomes. The so-called stealth domains in the α- and ß-subunit of GlcNAc-1-phosphotransferase were thought to be involved in substrate recognition and/or catalysis. Here, we performed in silico alignment analysis of stealth domain-containing phosphotransferases and showed that the amino acid residues Glu389 , Asp408 , His956 , and Arg986 are highly conserved between different phosphotransferases. Interestingly, mutations in these residues were identified in patients with MLII and MLIII alpha/beta. To further support the in silico findings, we also provide experimental data demonstrating that these four amino acid residues are strictly required for GlcNAc-1-phosphotransferase activity and thus may be directly involved in the enzymatic catalysis.

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis.

The amidinourea 8918 was recently reported to inhibit the type II phosphopantetheinyl transferase (PPTase) of Mycobacterium tuberculosis (Mtb), PptT, a potential drug-target that activates synthases and synthetases involved in cell wall biosynthesis and secondary metabolism. Surprisingly, high-level resistance to 8918 occurred in Mtb harboring mutations within the gene adjacent to pptT, rv2795c, highlighting the role of the encoded protein as a potentiator of the bactericidal action of the amidinourea. Those studies revealed that Rv2795c (PptH) is a phosphopantetheinyl (PpT) hydrolase, possessing activity antagonistic with respect to PptT. We have solved the crystal structure of Mtb's phosphopantetheinyl hydrolase, making it the first phosphopantetheinyl (carrier protein) hydrolase structurally characterized. The 2.5 Å structure revealed the hydrolases' four-layer (α/ß/ß/α) sandwich fold featuring a Mn-Fe binuclear center within the active site. A structural similarity search confirmed that PptH most closely resembles previously characterized metallophosphoesterases (MPEs), particularly within the vicinity of the active site, suggesting that it may utilize a similar catalytic mechanism. In addition, analysis of the structure has allowed for the rationalization of the previously reported PptH mutations associated with 8918-resistance. Notably, differences in the sequences and predicted structural characteristics of the PpT hydrolases PptH of Mtb and E. coli's acyl carrier protein hydrolase (AcpH) indicate that the two enzymes evolved convergently and therefore are representative of two distinct PpT hydrolase families.

Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions.

Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.

Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems.

OBJECTIVE: Fluorescent labeling of specific cell-surface proteins enables a manifold of techniques to study their function in health and disease. A frequently cited family of methods employs phosphopantetheinyl transferases (PPTases) to attach probes, provided as conjugates of Coenzyme A. This method appears attractive, as only short peptide tags genetically fused to the protein of interest are needed as conjugation sites. Here, we describe observations we made when evaluating such protocols for delicate single-molecule applications where we require a particular combination of dyes, low background binding or low labeling of other proteins, and a high degree of labeling. RESULTS: When we tested a PPTase-acceptor peptide couple with several experimental protocols and various CoA conjugates for labeling of a protein on the cell surface, we noticed substantial non-specific labeling. For the first time, we provide here a quantification of the non-specific fraction of the signals obtained using appropriate controls. We further present evidence that this background is due to CoA-dye conjugates entering the cell, where they may be covalently attached to endogenous proteins. However, when studying cell-surface proteins, most fluorescent readouts require that labeling is strictly limited to the protein of interest located at the cell surface. While such data have so far been missing in the literature, they suggest that for applications where labeling of unwanted molecules would affect the conclusions, researchers need to be aware of this potential non-specificity of PPTase methods when selecting a labeling strategy. We show, again by quantitative comparison, that the HaloTag is a viable alternative.

Investigation of the conserved reentrant membrane helix in the monotopic phosphoglycosyl transferase superfamily supports key molecular interactions with polyprenol phosphate substrates.

Long-chain polyprenol phosphates feature in membrane-associated glycoconjugate biosynthesis pathways across domains of life. These unique amphiphilic molecules are best known as substrates of polytopic membrane proteins, including polyprenol-phosphate phosphoglycosyl and glycosyl transferases, and as components of more complex substrates. The linear polyprenols are constrained by double bond geometry and lend themselves well to interactions with polytopic membrane proteins, in which multiple transmembrane helices form a rich landscape for interactions. Recently, a new superfamily of monotopic phosphoglycosyl transferase enzymes has been identified that interacts with polyprenol phosphate substrates via a single reentrant membrane helix. Intriguingly, despite the dramatic differences in their membrane-interaction domains, both polytopic and monotopic enzymes similarly favor a unique cis/trans geometry in their polyprenol phosphate substrates. Herein, we present a multipronged biochemical and biophysical study of PglC, a monotopic phosphoglycosyl transferase that catalyzes the first membrane-committed step in N-linked glycoprotein biosynthesis in Campylobacter jejuni. We probe the significance of polyprenol phosphate geometry both in mediating substrate binding to PglC and in modulating the local membrane environment. Geometry is found to be important for binding to PglC; a conserved proline residue in the reentrant membrane helix is determined to drive polyprenol phosphate recognition and specificity. Pyrene fluorescence studies show that polyprenol phosphates at physiologically-relevant levels increase the disorder of the local lipid bilayer; however, this effect is confined to polyprenol phosphates with specific isoprene geometries. The molecular insights from this study may shed new light on the interactions of polyprenol phosphates with diverse membrane-associated proteins in glycoconjugate biosynthesis.