Ternary Complex Mechanism Research Articles

Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

Mutational and structural studies of (βα)8-barrel fold methylene-tetrahydropterin reductases utilizing a common catalytic mechanism.

Methylene-tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one-carbon (C1) metabolisms. F420-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) reductase (Mer) and the flavin-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD-dependent methylene-H4F reductase (MTHFR) uses FAD as prosthetic group and a ping-pong mechanism to catalyze the reduction of methylene-H4F. A ternary complex structure and a thereof derived catalytic mechanism of MTHFR is available, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F420 were determined. A ternary complex of jMer was modeled on the basis of the jMer-F420 structure and the ternary complex structure of MTHFR by superimposing the polypeptide after fixing hydride-transferring atoms of the flavins on each other, and by the subsequent transfer of the methyl-tetrahydropterin from MTHFR to jMer. Mutational analysis of four functional amino acids, which are similarly positioned in the three reductase structures, indicated despite the insignificant sequence identity, a common catalytic mechanism with a 5-iminium cation of methylene-tetrahydropterin as intermediate protonated by a shared glutamate. According to structural, mutational and phylogenetic analysis, the evolution of the three reductases most likely proceeds via a convergent development although a divergent scenario requiring drastic structural changes of the common ancestor cannot be completely ruled out.

Simultaneous geraniol and citronellol transesterification using Pseudomonas fluorescens lipase for the production of fragrance and flavour esters: A kinetic study

This study aims to develop, for the very first time, a kinetic model for the simultaneous transesterification of geraniol and citronellol using Pseudomonas fluorescens lipase to produce their respective esters. This allows us to test the feasibility of the bioprocess and comprehend the enzyme behaviour, fine-tune process efficiency, and predict process performance. Geranyl and citronellyl esters are key ingredients in fragrance and flavour formulations in the food sector, as well as bioactive compounds for pest and insect repellents in the agricultural sector. Unlocking a greener and sustainable production of these terpene esters directly from plant-derived essential oil is crucial to meet the 2050 net zero targets to produce bio-based chemicals. The simultaneous transesterification of geraniol and citronellol in a batch reactor successfully achieved conversions of 66 % for citronellol and 94 % for geraniol within 5 h. Key optimum conditions were 1:8 molar ratio (geraniol:vinyl acetate), an enzyme loading of 10 wt % of geraniol, temperature of 40 °C, and a stirring speed of 250 RPM. The results indicated that simultaneous transesterification followed a ternary complex mechanism with competitive inhibition by alcohol. Initial testing of rose geranium oil under optimised conditions yielded 32 % citronellol and 50 % geraniol conversion after 30 h. Overall, this study shows promise as a green pathway utilising enzyme catalysis to selectively produce esters from diverse terpene mixtures and can be extended to terpene alcohols derived from flower essential oils.

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway.

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.

SARM1, an Enzyme Involved in Axon Degeneration, Catalyzes Multiple Activities through a Ternary Complex Mechanism.

Sterile alpha and toll/interleukin receptor (TIR) motif containing protein 1 (SARM1) is an NAD+ hydrolase and cyclase involved in axonal degeneration. In addition to NAD+ hydrolysis and cyclization, SARM1 catalyzes a base exchange reaction between nicotinic acid (NA) and NADP+ to generate NAADP, which is a potent calcium signaling molecule. Herein, we describe efforts to characterize the hydrolysis, cyclization, and base exchange activities of TIR-1, the Caenorhabditis elegans ortholog of SARM1; TIR-1 also catalyzes NAD(P)+ hydrolysis and/or cyclization and regulates axonal degeneration in worms. We show that the catalytic domain of TIR-1 undergoes a liquid-to-solid phase transition that regulates not only the hydrolysis and cyclization reactions but also the base exchange reaction. We define the substrate specificities of the reactions, demonstrate that cyclization and base exchange reactions occur within the same pH range, and establish that TIR-1 uses a ternary complex mechanism. Overall, our findings will aid drug discovery efforts and provide insight into the mechanism of recently described inhibitors.

Crystal structure of FAD-independent methylene-tetrahydrofolate reductase from Mycobacterium hassiacum.

FAD-independent methylene-tetrahydrofolate (methylene-H4 F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H4 F to methyl-H4 F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H4 F reductase (MTHFR), although the latter uses a ping-pong mechanism with the prosthetic group as intermediate hydride carrier. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR encoding gene. Therefore, Mfr appears to be an excellent target for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization, and the crystal structure of Mfr from the thermophilic mycobacterium Mycobacterium hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR have minor sequence identity and different catalytic mechanisms, their structures are highly similar, thus suggesting a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both hMfr and MTHFR. Thus, highly similar binding modes for the C1 -carriers and the reducing agents in hMfr and MTHFR are assumed.

Stabilization of a collagen peptide-cranberry juice by three functional polysaccharides with different charge characteristics

Preventing the precipitation of collagen peptide-fruit juice drinks during production and storage is a current challenge. This study investigated the efficiency and mechanism of β-glucan, low methoxyl pectin, and chitosan (a neutral, anionic, and cationic polysaccharide, respectively) for enhancing the stability of a fish collagen peptide-cranberry juice drink. The effects of mixing sequence, mixing temperature, pH, and polysaccharide concentration on stability were studied by conducting centrifugal precipitation rate, turbidity, relative turbidity, zeta potential, rheology, confocal laser scanning microscopy, and storage (4 °C, 25 °C, 40 °C, 14 d) experiments. All three polysaccharides effectively improved the stability of the drink mixture, but to different extents, as the stability was highly dependent on pH and the type and concentration of the polysaccharide. Steric hindrance, electrostatic repulsion, and increased viscosity were the main reasons underlying the improvements in the stability of the system. The stability mechanisms were also related to a competition or ternary complex mechanism. The results of storage experiments showed that chitosan could only temporarily stabilize system (25 °C, 2d), whereas β-glucan (4 °C, 14d) and low-methoxyl pectin (4 °C, 25 °C, 14d) provided the continuous stability. Overall, these results can provide important guidance for the application of these three polysaccharide types in the beverage industry to develop and select ideal formulations for collagen peptide-juice drinks.

Kinetics and mechanisms of catalyzed dual-E (antithetic) controllers.

Homeostasis plays a central role in our understanding how cells and organisms are able to oppose environmental disturbances and thereby maintain an internal stability. During the last two decades there has been an increased interest in using control engineering methods, especially integral control, in the analysis and design of homeostatic networks. Several reaction kinetic mechanisms have been discovered which lead to integral control. In two of them integral control is achieved, either by the removal of a single control species E by zero-order kinetics (“single-E controllers”), or by the removal of two control species by second-order kinetics (“antithetic or dual-E control”). In this paper we show results when the control species E1 and E2 in antithetic control are removed enzymatically by ping-pong or ternary-complex mechanisms. Our findings show that enzyme-catalyzed dual-E controllers can work in two control modes. In one mode, one of the two control species is active, but requires zero-order kinetics in its removal. In the other mode, both controller species are active and both are removed enzymatically. Conditions for the two control modes are put forward and biochemical examples with the structure of enzyme-catalyzed dual-E controllers are discussed.

The ArsH Protein Product of the Paracoccus denitrificans ars Operon Has an Activity of Organoarsenic Reductase and Is Regulated by a Redox-Responsive Repressor.

Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.

Crystal structure of Tam41 cytidine diphosphate diacylglycerol synthase from a Firmicutes bacterium.

Translocator assembly and maintenance 41 (Tam41) catalyses the synthesis of cytidine diphosphate diacylglycerol (CDP-DAG), which is a high-energy intermediate phospholipid critical for generating cardiolipin in mitochondria. Although Tam41 is present almost exclusively in eukaryotic cells, a Firmicutes bacterium contains the gene encoding Tam41-type CDP-DAG synthase (FbTam41). FbTam41 converted phosphatidic acid (PA) to CDP-DAG using a ternary complex mechanism in vitro. Additionally, FbTam41 functionally substituted yeast Tam41 in vivo. These results demonstrate that Tam41-type CDP-DAG synthase functions in some prokaryotic cells. We determined the crystal structure of FbTam41 lacking the C-terminal 18 residues in the cytidine triphosphate (CTP)-Mg2+ bound form at a resolution of 2.6Å. The crystal structure showed that FbTam41 contained a positively charged pocket that specifically accommodated CTP-Mg2+ and PA in close proximity. By using this structure, we constructed a model for the full-length structure of FbTam41 containing the last a-helix, which was missing in the crystal structure. Based on this model, we propose a molecular mechanism for CDP-DAG synthesis in bacterial cells and mitochondria.

Chemoenzymatic Epoxidation of Limonene Using a Novel Surface-Functionalized Silica Catalyst Derived from Agricultural Waste.

Limonene is one of the most important terpenes having wide applications in food and fragrance industries. The epoxide of limonene, limonene oxide, finds important applications as a versatile synthetic intermediate in the chemical industry. Therefore, attempts have been made to synthesize limonene oxide using eco-friendly processes because of stringent regulations on its production. In this regard, we have attempted to synthesize it using a cost-effective and eco-friendly process. Chemoenzymatic epoxidation of limonene to limonene oxide was carried out using in situ generation of peroxy octanoic acid from octanoic acid and H2O2. In this study, agricultural-waste rice husk ash (RHA)-derived silica was surface-functionalized using (3-aminopropyl) triethoxysilane (APTS), which was cross-linked using glutaraldehyde for immobilization of Candida antarctica lipase B. Furthermore, the immobilized enzyme was entrapped in calcium alginate beads to avoid enzyme leaching. Thus, limonene oxide was prepared using this catalyst under conventional and microwave heating. The microwave irradiation intensifies the process, reducing the reaction time under the same conditions. Maximum conversion of limonene to limonene oxide of 75.35 ± 0.98% was obtained in 2 h at 50 °C using a microwave power of 50 W. In the absence of microwave irradiation, the conventional heating gave 44.6 ± 1.14% conversion in 12 h. The reaction mechanism was studied using the Lineweaver–Burk plot, which follows a ternary complex mechanism with inhibition due to peroxyoctanoic acid (in other words H2O2). The prepared catalyst shows high reusability and operational stability up to four cycles.

Optimization of Enzymatic Synthesis of Neryl Acetate in a Solvent Free System

Synthesis of valuable flavour and fragrance compounds is one of the intense applications of lipase-catalysed transesterification. This paper demonstrated application of Novozyme 435 to the synthesis of neryl acetate in a solvent free system. Response surface methodology (RSM) complemented by central com-posite design (CCD) was used to evaluate the effect of various process param-eters on reaction conversion. The results showed that 91.6% conversion of nerol with 100% selectivity to neryl acetate was obtained under the conditions of 1:12.6 mole ratio of nerol to ethyl acetate, 2.6% (w/w) Novozyme 435 at 52.7?C for 2 h. From the kinetic study, it was verified that the reaction followed a ternary complex mechanism with inhibition by ethyl acetate. The process was green, clean and useful to the perfume and flavour industries.

Enhancing Activity by Supercritical CO2 Mediated Immobilization of Lipase on Mesocellular Foam in Preparation of Hexyl Laurate.

Hexyl laurate is employed in several cosmetics having great demand. It could be synthesized catalytically like a "natural" perfume using a lipase. The use of mesocellular foam silica (MCF) for immobilization of lipases could be made using supercritical CO2 as a medium to enhance its activity in comparison with the normal techniques. Three different catalysts were supported on MCF such as Candida antractica B (CALB), Amano AYS, and Porcine pancreas (PPL), and their activity was evaluated in the preparation of hexyl laurate from lauric acid and hexyl alcohol. CALB@ MCF was the best among all. A systematic study was conducted to assess the effects of different operating parameters. It was ternary complex mechanism with inhibition by hexyl alcohol. The enzyme was reusable and the process is green.

Surface functionalization of SBA-15 for immobilization of lipase and its application in synthesis of alkyl levulinates: Optimization and kinetics

Abstract Levulinic acid, being one the most important building block precursors, is of prime importance in a variety of industries. The esters of levulinic acid have significant applications in fields of flavor and fragrance. In this study, surface functionalized SBA-15 was used to immobilize Candida antarctica lipase B for the production of different alkyl levulinates. APTS (3-aminopropyl) triethoxysilane functionalized SBA-15 was used to immobilize lipase (Lip) cross-linked with GLU (glutaraldehyde) (named SBA-15-APTS-GLU-Lip). Various aspects of the immobilized catalyst were studied including surface area analysis, morphology, stability using XRD, SEM, TEM, and FTIR techniques. Process optimization of the model reaction of levulinic acid and isoamyl alcohol was done for the production of isoamyl levulinate. The highest conversion of 94% was achieved within 2 h at 50 °C. The kinetics was found to obey ternary dead end complex mechanism with isoamyl alcohol inhibition. The results indicate that the synthesized biocatalyst shows high operational stability and reusability up to 4 cycles.

Process intensification by microwave irradiation in immobilized-lipase catalysis in solvent-free synthesis of ethyl valerate

Valeric esters are a promising energy source and used in different industries ranging from biofuels, perfumes, food, flavors to pharmaceuticals. Ethyl valerate is one of the esters possessing all these attributes as an industrial chemical. In the current work an enzymatic route was examined using valeric acid and ethanol with commercially available lipases, namely, Lipozyme RM IM, Lipozyme TL IM and Novozyme 435 (Candida antarctica lipase B) under solvent fee condition and microwave irradiation. Novozym 435 could lead to 69.2% conversion of valeric acid at 50 °C using equimolar ratio of valeric acid and ethanol at enzyme loading of 10.06 g L−1 in 40 min under microwave irradiation. Lineweaver-Burk plot shows ternary complex mechanism with inhibition by ethanol. A reaction mechanism is proposed and kinetic rate equation derived. The enzyme is reusable.

A Green Process for Synthesis of Geraniol Esters by Immobilized Lipase from Candida Antarctica B Fraction in Non-Aqueous Reaction Media: Optimization and Kinetic Modeling

Abstract Enzymatic synthesis of molecules such as flavors, perfumes and fragrances has a great commercial advantage of being marketed as “natural” and also it offers exquisite selectivity of enzymes that can be superior over chemical catalysis. The current work focuses on the enzymatic synthesis of geranyl acetate as model compound, including optimization of reaction conditions such as nature of catalyst, reaction media, speed of agitation, mole ratio and temperature. A variety of esters were also synthesized. Geraniol was esterified with various acids, aromatic esters and vinyl esters in 1:4 molar ratio. Among all vinyl ester was the best giving in good yield (77–100 %) as compared to aromatic esters (5–82 %) and acids (7–31 %). Novozym 435 was found to be most active catalyst with ~96 % conversion and 100 % selectivity in 60 min at 55 °C in n-heptane as solvent for geranyl acetate. The maximum reaction rate was estimated (Vmax = 0.2712 mol L−1 min-1) by using the double reciprocal plot. It is a ternary complex (ordered bi-bi) mechanism with inhibition by geraniol.

Microwave assisted solvent-free synthesis of n -butyl propionate by immobilized lipase as catalyst

Abstract Operational simplicity, solvent free media, potential reusability of catalyst and wide functional group tolerance are the advantages for solvent free synthesis under microwave irradiation technique. In the current work, n-butyl propionate was synthesized by esterification of propionic acid with n-butanol using different immobilized enzymes under microwave irradiation vis-a-vis conventional heating. Commercially available lipases such as Lypozyme RM IM, Lypozyme TLIM and Novozym 435 (Candida antarctica lipase B) were screened for n-butyl propionate synthesis from propionic acid and n-butanol for which Novozym 435 showed the best catalytic activity. n-Butyl propionate is widely used in flavour, painting, enamels, appliance coating and similar processes. In 8 h 92% conversion was achieved at 60 °C with 1:12 mol ratio of propionic acid to n-butanol under microwave irradiation. Systematic studies were done to understand mechanism and kinetics of reaction. By studying the Lineweaver-Burk plots it was found that the lines were intersecting at a point, showing that the reaction follows ternary complex mechanism with inhibition by n-butanol.

Steady-state analysis of enzymes with non-Michaelis-Menten kinetics: The transport mechanism of Na+/K+-ATPase

Procedures to define kinetic mechanisms from catalytic activity measurements that obey the Michaelis-Menten equation are well established. In contrast, analytical tools for enzymes displaying non-Michaelis-Menten kinetics are underdeveloped, and transient-state measurements, when feasible, are therefore preferred in kinetic studies. Of note, transient-state determinations evaluate only partial reactions, and these might not participate in the reaction cycle. Here, we provide a general procedure to characterize kinetic mechanisms from steady-state determinations. We described non-Michaelis-Menten kinetics with equations containing parameters equivalent to kcat and Km and modeled the underlying mechanism by an approach similar to that used under Michaelis-Menten kinetics. The procedure enabled us to evaluate whether Na+/K+-ATPase uses the same sites to alternatively transport Na+ and K+ This ping-pong mechanism is supported by transient-state studies but contradicted to date by steady-state analyses claiming that the release of one cationic species as product requires the binding of the other (ternary-complex mechanism). To derive robust conclusions about the Na+/K+-ATPase transport mechanism, we did not rely on ATPase activity measurements alone. During the catalytic cycle, the transported cations become transitorily occluded (i.e. trapped within the enzyme). We employed radioactive isotopes to quantify occluded cations under steady-state conditions. We replaced K+ with Rb+ because 42K+ has a short half-life, and previous studies showed that K+- and Rb+-occluded reaction intermediates are similar. We derived conclusions regarding the rate of Rb+ deocclusion that were verified by direct measurements. Our results validated the ping-pong mechanism and proved that Rb+ deocclusion is accelerated when Na+ binds to an allosteric, nonspecific site, leading to a 2-fold increase in ATPase activity.

Synthesis of Geraniol Esters in a Continuous-Flow Packed-Bed Reactor of Immobilized Lipase: Optimization of Process Parameters and Kinetic Modeling.

With increasing demand for perfumes, flavors, beverages, and pharmaceuticals, the various associated industries are resorting to different approaches to enhance yields of desired compounds. The use of fixed-bed biocatalytic reactors in some of the processes for making fine chemicals will be of great value because the reaction times could be reduced substantially as well as high conversion and yields obtained. In the current study, a continuous-flow packed-bed reactor of immobilized Candida antarctica lipase B (Novozym 435) was employed for synthesis of various geraniol esters. Optimization of process parameters such as biocatalyst screening, effect of solvent, mole ratio, temperature and acyl donors was studied in a continuous-flow packed-bed reactor. Maximum conversion of ~87% of geranyl propionate was achieved in 15min residence time at 70°C using geraniol and propionic acid with a 1:1mol ratio. Novozym 435 was found to be the most active and stable biocatalyst among all tested. Ternary complex mechanism with propionic acid inhibition was found to fit the data.

Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate

Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs.