Phosphoglycosyl transferase C (PglC) is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

belonging to a class known as monotopic phosphoglycosyl transferases (PGT). PGTs are required for the synthesis of

glycoconjugate In molecular biology and biochemistry, glycoconjugates are the classification family for carbohydrates – referred to as glycans – which are covalently linked with chemical species such as proteins, peptides, lipids, and other compounds. Glyc ...

s on the membrane surface of bacteria''.'' Glycoconjugates, such as glycoproteins, are imperative for bacterial communication as well as host cell interactions between

prokaryotic A prokaryote (; less commonly spelled procaryote) is a single-celled organism whose cell lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Greek (), meaning 'before', and (), meaning 'nut' ...

and

eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...

cells lending to bacteria's pathogenicity.

Background

PglC is found in the pathogenic gram-negative organism ''

Campylobacter jejuni ''Campylobacter jejuni'' is a species of pathogenic bacteria that is commonly associated with poultry, and is also often found in animal feces. This species of microbe is one of the most common causes of food poisoning in Europe and in the US, w ...

'' (''C. jejuni'')''.'' Infection from ''C. jejuni'' results in acute gastroenteritis followed by vomiting, diarrhea, fever and abdominal pain. The most common route of infection is through undercooked poultry as birds are a common source of ''C. jejuni''. Recent studies have also shown an association between prior ''C. jejuni'' infection and the neurological syndrome Guillan-Barré. The glycoconjugates,

lipopolysaccharide Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...

s (LPS), found in the membrane of the bacteria resemble

ganglioside A ganglioside is a molecule composed of a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids (e.g. N-acetylneuraminic acid, ''N''-acetylneuraminic acid, NANA) linked on the sugar chain. NeuNAc, an acetylated derivative ...

s found in the human nervous system leading to the generation of autoantibodies which cause deterioration of neurons. Gangliosides can be found on neuronal cells and are membrane proteins that aid in cell-cell recognition and communication. PglC belongs to a superfamily of enzymes known as monotopic phosphoglycsoyl transferases (monoPGT). These membrane-associated proteins catalyze the transfer of a phosphosugar from a soluble

nucleoside Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotid ...

diphosphate-activated donor to a polyprenol phosphate (Pren-P) acceptor within the membrane. The product is then diversified via action of glycosyl transferases, to build a lipid linked oligosaccharide that will be flipped to the periplasm and form a glycoconjugate. Mono PGTs are unique to prokaryotes and essential for the production of glycoconjugates which mediate cell-host interactions during bacterial infections and are thus important for bacterial survival and pathogenicity. Glycoconjugates are integral structures on the surface of cell membranes composed of carbohydrates linked to other biomolecules such as proteins or lipids. These structures serve as a shield to the environment as well as aid in pathogenesis and viability of the bacterium itself. Glycoconjugates are also known to comprise

adhesins Bacterial adhesins are cell-surface components or appendages of bacteria that facilitate adhesion or adherence to other cells or to surfaces, usually in the host they are infecting or living in. Adhesins are a type of virulence factor. Adherence is ...

used for host colonization and invasion. ''C. jejuni'' utilizes adhesins to attach to the epithelial cells of the gastrointestinal tract of humans allowing for colonization and infection of the human host. Eukaryotic cells exhibit their own glycoconjugate ecosystem lending to immune system recognition of human cells as "self". Bacteria utilize glycan

mimicry In evolutionary biology, mimicry is an evolved resemblance between an organism and another object, often an organism of another species. Mimicry may evolve between different species, or between individuals of the same species. In the simples ...

to pose as eukaryotic cells and evade immune response. Several studies have shown that inactivated genes linked to glycan synthesis result in an inability of bacteria to adhere to host cells thereby inhibiting colonization of the host. PglC_5W7L

Structure

PglC is a membrane protein which only enters the first leaflet of the membrane on the cytosolic side of the lipid membrane interface. That is, the protein only sits in the first layer of the double-layered membrane. PglC from ''Campylobacter jejuni'' has yet to be structurally characterized, but an

orthologue Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a speci ...

of PglC from ''Campylobacter concisus'' was elucidated in 2018 that represents the minimal functional core of this class of proteins. Primary structure and

hidden markov model A hidden Markov model (HMM) is a Markov model in which the observations are dependent on a latent (or ''hidden'') Markov process (referred to as X). An HMM requires that there be an observable process Y whose outcomes depend on the outcomes of X ...

computational analysis had actually predicted PglC to be a bitopic membrane protein. A bitopic membrane protein is one that passes through both layers of the membrane, but only does so once. However, structural characterization revealed that PglC only pans the first leaflet of the membrane. The structure also revealed significant characteristics of the protein important to its function such as the reentrant membrane helix (blue/light blue) that dips into the first leaflet of the membrane as well as the highly conserved Asp-Glu catalytic dyad within the active site (green loop). The active site also holds a phosphate binding site and Mg²⁺ cofactor site important for coordinating reaction chemistry. The other helices exist at the membrane interface (red, green, orange).

Enzyme superfamily

Membrane proteins exist in three topologies: polytopic, bitopic, and monotopic, depending on the distribution of their domains throughout the membrane. The domains of polytopic proteins cross the membrane multiple times while bitopic proteins may only pass through the membrane once typically with a

transmembrane helix A transmembrane domain (TMD, TM domain) is a membrane-spanning protein domain. TMDs may consist of one or several alpha-helices or a transmembrane beta barrel. Because the interior of the lipid bilayer is hydrophobic, the amino acid residues in T ...

connecting two soluble domains outside of the membrane. Monotopic membranes make up the smallest percentage of membrane proteins (0.06%) and are embedded in a single layer of the membrane, not both. While the topologies of bitopic and polytopic membrane proteins can be linked to their function, monotopic proteins' topologies have yet to inform any function of these unique proteins apart from commonly being found in pathways where several enzymes are localized in sequence within the membrane. Monotopic_vs_polytopic

PglC is the first structurally characterized member of the monotopic PGT

superfamily SUPERFAMILY is a database and search platform of structural and functional annotation for all proteins and genomes. It classifies amino acid sequences into known structural domains, especially into SCOP superfamilies. Domains are functional, str ...

. Other PGT superfamilies include the polytopic PGTs which are commonly exemplified by the proteins MraY and WecA. Although the three PGTs share the same function, they differ in structure and mechanism. PglC features a reentrant membrane helix that only spans the first leaflet of the membrane while MraY has multiple transmembrane helices. The mechanisms by which the two superfamilies' catalyze addition of an NDP-sugar (nucleoside di-phosphate) to a polyprenol acceptor within the membrane are unique. PglC utilizes a two-step

ping pong mechanism Enzyme kinetics is the study of the rates of enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in th ...

that generates a covalent intermediate which is then available for

nucleophilic In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they a ...

attack by the polyprenol acceptor within the membrane. MraY and other polytopic PGTs use a

ternary complex A ternary complex is a protein complex containing three different molecules that are bound together. In structural biology, ''ternary complex'' can also be used to describe a crystal containing a protein with two small molecules bound, such as a ...

mechanism whereby Pren-P and the NDP-sugar are reacted by enzyme within a single step.

Function

PglC is involved in the first membrane-associated

catalysis Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...

step involved in the synthesis of glycoconjugates in the bacterium ''Campylobacter jejuni''. The substrate for PglC, UDP-di-N-acetyl-bacillosamine, is first synthesized in the cytosol by the enzymes PglD, PglE, and PglF. PglC is responsible for linking the sugar, di-N-acetyl-bacillosamine, to the polyprenol phosphate (pren-p) acceptor within the membrane. PglC is also unique compared to its successors in the pathway due to the attachment of a phosphosugar to pren-p. PglA, PglJ, PglH, PglI, PglK, PglB are known as glycosyltransferases and each add their own respective sugars to the growing glycan, but there is no addition of a phosphoryl group. The phosphoryl group is integral for initiation of the membrane-associated part of the pathway, otherwise no successive sugars can be added to the growing glycan.

Mechanism

PglC catalyzes the generation of a polyprenol diphoshate-linked sugar ( bacillosamine) via a

. The AspGlu catalytic dyad of PglC acts as a nucleophile to attack UDP-bacillosamine, releasing UMP in the process (step 1). A covalent intermediate characteristic of a ping pong mechanism is formed between the enzyme and the sugar via the phosphate group. Polyprenol phosphate (Pren-P), a membrane substrate, attacks the covalent intermediate, or phosphoglycosyl

adduct In chemistry, an adduct (; alternatively, a contraction of "addition product") is a product of a direct addition of two or more distinct molecules, resulting in a single reaction product containing all atoms of all components. The resultant is ...

, resulting in turnover of enzyme and attachment of the sugar to the Pren-P acceptor within the membrane.

References

External links

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