Cis-Prenyltransferases (cis-PTs) is a large enzyme family which is well conserved in all domains of life. Cis-PTs catalyze condensation reactions of isopentenyl pyrophosphate (IPP) and produce linear polyprenyl diphosphate. The length of this isoprenoid carbon chain varies from short molecules like geranyl diphosphate (C10) to natural rubber (C>10’000). The human cis-Prenyltransferase Complex (hcis-PT) has an essential role in protein N-glycosylation. It synthesises the precursor of glycosyl carrier dolichol-phosphate. Mutations in genes coding for hcis-PT can cause severe diseases, such as retinitis pigmentosa.

Structure

The hcis-PT subunits

The hcis-PT is a tetramer formed by assembling a dimer of heterodimers. The hcis-PT is composed of two catalytically active dehydrodolichyl diphosphate synthases (DHDDS) and the Nogo-B receptor (NgBR). The NgBR can be divided 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 motif which might have an important role in hcis-PT activity. The structure of DHDDS can be divided into three domains: C-terminal domain (residues 251–333), an N-terminal domain (residues 1–26) and a canonical catalytic cis-PT homology domain (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 directed into 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. In fact, NgBR active site does not have a visible substrate-binding cavity. The 3 β-strands and 2 α-helices are packed via hydrophobic interactions so there is no cavity anymore 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 formed by a superficial polar region stabilizing the interaction between IPP and a deep hydrophobic tunnel which accommodate the elongating carbon chain. In the active-site, there are two substrate-binding sites, a S1 and a S2 site. The S1 site binds the initiatory substrate FPP. It also interacts with Mg²⁺ ions which are crucial for IPP hydrolysis during the condensation reaction. The Mg²⁺ is octahedrally coordinated and stabilized. S2 site binds the IPP molecule which will be used for chain elongation. The C-terminus of NgBR (RxG) is directly involved in forming the superficial polar region and enable the formation of S1 and S2. In fact, at the S1 site, we have two polar interaction networks between NgBR and DHDDS. At S2 site, the backbone nitrogen atoms directly coordinate the phosphate molecule.

The elongation reaction in the hydrophobic active-site tunnel

The cis-PT catalyze 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 deep hydrophobic tunnel. First, the pyrophosphate group (FPP) of the initiatory substrate, which interacts with a Mg²⁺ ion, is hydrolyzed at the S1 site. Then, the condensation of remaining carbon with the IPP from S2 site follows. The elongated products translocate to the S1 so the carbon chain goes into the hydrophobic tunnel of the active site. At 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 larger in DHDDS compared to short and medium chain cis-PT. The larger the diameter, the better is the accommodation of longer product.

Importance of the enzyme

Product of the N-glycosylation

Disease comprehension

The Retinitis pigmentosa is a hereditary disease which consist in the human retina degeneration. The patient loose progressively a part of his sight: night vision when he is a teenager, side vision when he is a young adult and finally central vision in later life. This loss of sight is due to the progressive loss of rod and cone photoreceptor cells which provide coloured vision. Scientists also observed, thanks to electroretinogram, 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, which means that 40% of cases of Retinitis pigmentosa diseases are due to unidentified genes [5]. Concerning treatments, there is today no possible cure, even if some trials showed a slow down of the disease with the consumption of vitamin A palmitate foods and omega-3-rich fish [5].

Structural highlights

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