Cis-Prenyltransferases (cis-PTs) is a large enzyme family which is well conserved in all areas of life. Cis-PTs catalyze condensation reactions of isopentenyl pyrophosphate (IPP) and produce linear polyprenyl diphosphate. The length of this isoprenoid carbon chain ranges from short molecules like geranyl diphosphate (C10) to natural rubber (C>10’000). The human cis-Prenyltransferase Complex (hcis-PT) plays an essential role in protein N-glycosylation. It synthesizes the precursor of glycosyl carrier dolichol-phosphate. Mutations in genes coding for hcis-PT can cause serious diseases, such as retinitis pigmentosa.[1]
Structure
The hcis-PT subunits
The hcis-PT is a tetramer formed by the assembly of a dimer of heterodimers. The hcis-PT is composed of two catalytically active dehydrodolichyl diphosphate synthases () and the Nogo-B receptor (). The NgBR can be split into two domains: a C-terminal pseudo prenyltransferase domain that interacts with DHDDS and an N-terminal transmembrane domain that is anchored in the endoplasmic reticulum. The NgBR C-terminus encompasses the RxG pattern which might have an important role in hcis-PT activity and which is not present in NUS1 (6jcn), the yeast homolog of NgBR. The structure of DHDDS can be divided into three domains: a (residues 251–333), a (residues 1–26) and a (residues 27–250). The cis-PT homology domain heterodimerizes with NgBR. The tetramer is formed by heterotypic interactions of the "turn" region with NgBR and by homotypic interactions between DHDDS. The whole complex is headed towards the cytosol.
The active site organization
Farnesyl diphosphate (FPP) and Mg2+ are only detected in the active site of DHDDS and only the active site of DHDDS is required for the catalytic activity. NgBR induces an increase in the expression and activity of the complex, but itself has no catalytic activity. Indeed, NgBR active site does not have a visible substrate-binding cavity. The 3 β-strands and 2 α-helices are packaged via hydrophobic interactions, so there is no longer a cavity and it is devoid of water.
Catalytical activity of the human cis-prenyltransferases
The catalytical domain of DHDDS is homologous to undecaprenyl pyrophosphate synthase (UPPS) with 2 α-helices and four β-strands within each monomer. The active site is shaped by a that stabilizes the interaction between IPP and a which accommodate the elongating carbon chain. In the active site, there are two substrate-binding sites, an S1, and an S2 site. The binds the initiator substrate FPP. It additionally interacts with Mg2+ ions which are crucial for IPP hydrolysis during the condensation reaction. The Mg2+ is stabilized and by three surrounding water molecules, two oxygens of the pyrophosphate and one carboxylate oxygen of D34, that ensures the biological function of the protein. binds the IPP molecule which will be employed for chain elongation. The C-terminus of NgBR (RxG) is directly involved in forming the superficial polar layer and enables the formation of S1 and S2. In fact, at the S1 site, there are two polar interaction networks between NgBR and DHDDS. At , the backbone nitrogen atoms directly coordinate the phosphate molecule.
The elongation reaction in the hydrophobic active-site tunnel
The cis-PT catalyzes the chain elongation of FPP by successive head-to-tail condensation with a specific number of IPP to form linear lipids with designated chain lengths. The reaction is as follows:
IPP headgroups are bound to the enzyme at the superficial polar region while the carbon chains point toward the . First, the pyrophosphate group (FPP) of the initiatory substrate, which interacts with a Mg2+ ion, is hydrolyzed at the . Then, the condensation of the remaining carbon with the IPP from the S2 site follows. The elongated product translocate to the S1, so the carbon chain goes into the hydrophobic tunnel of the active site. In the end, a new IPP molecule binds to the S2 site and the cycle is repeated until the active site can no longer accommodate the long-chain isoprenoid.
Regulation of the product length
The hydrophobic tunnel of DHDDS is formed by 2 α-helix and 4 β-strands. The opening between the 2 α-helices is larger in DHDDS compared to short and medium-chain cis-PT. The larger the diameter, the better is the accommodation of a longer product.
Functional importance and link to human health
Product of the N-glycosylation
The N-glycosylationis a post-translational modification realized in the endoplasmic reticulum of the cell. This process consists in linking a glycan to a protein, which provide the biological protein fonction and folding. The hcis-PT produces DHDD (dehydrodolichyl diphosphate), an important precursor molecule for the dolichol-phosphate lipid carrier needed in the N-glycosylation reaction.
Disease comprehension
The Retinitis pigmentosa is a hereditary disease. The patient loses progressively a part of his sight: night vision as a teenager, side vision as a young adult, and central vision in later life. This loss of sight is due to the progressive decrease of rod and cone photoreceptor cells which provide colored vision. Scientists observed that symptoms appear many years after the beginning of the photoreceptor’s degeneration in most cases. Retinitis pigmentosa can be encoded by many genes. More than 45 have been identified but these genes concern only 60% of sick patients. Therefore 40% of cases of Retinitis pigmentosa are unidentified genes. Until today there is no cure. However, studies showed a slow down of the disease with the intake of vitamin A, palmitate foods and omega-3-rich fish [2].
For instance, missense mutations can provoke an autosomal recessive Retinitis pigmentosa (arRP). These mutations concern the S1 and S2 sites of the active site where pyrophosphate can bind and most of the mutations related to the disease impact directly the substrate binding, according to scientists. For one mutation, , it is more complicated. Scientists remarked that, in the (short distance). The mutation K42E provokes in the protein scale, hypothetically, an interaction with the adjacent active-site residues R38 positively charged. So R38 points away from the active site cavity, forming a new stable salt bridge with the mutant E42. The distance between K42E and E234 is longer due to the charge repulsion. Finally, experiments proved that aberrant polar networks are due to the K42E mutation, disturbing the active-site residues which can’t interact with the substrate and leading to a decrease of the catalytic activity [1].
References
[1] Michal Lisnyansky Bar-El et al. «Structural basis of heterotetrameric assembly and disease mutations in the human cis-prenyltransferase ». Nature Communications. 11:523, (2020).
[2 ]Dyonne T Hartong et al. « Retinitis Pigmentosa ». The Lancet. 18;368(9549):1795‑809, (2006)
