Structural Insights into Heterodimerization and Catalysis of the Human Cis- prenyltransferase “NgBR/DHDDS” Complex

Cis-prenyltransferses (cis-PTases) constitute a family of enzymes involved in the synthesis of isoprenoid lipids required for various biological functions across all domains of life. The eukaryotic cis-PTase catalyzes the rate-limiting step in the synthesis of dolichyl phosphate, an indispensable glycosyl carrier lipid required for protein glycosylation in the lumen of endoplasmic reticulum. Based on enzyme composition, cis-PTases can be either homomeric or heteromeric enzymes. The human cis-PTase possesses a heteromeric configuration consisting of the two evolutionary related subunits: NgBR (dehydrodolichyl diphosphate synthase accessory subunit, first identified as a Nogo-B receptor) and DHDDS (dehydrodolichyl diphosphate synthase catalytic subunit). Recently, several mutations in both subunits have been reported to associate with various human diseases, collectively known as congenital disorders of glycosylation (CDG), including severe CDG type I, developmental and epileptic encephalopathy, and autosomal recessive retinitis pigmentosa. In addition, mutations on the NgBR subunit have been recently reported in patients suffering from early onset of Parkinson’s disease (EOPD). Despite its crucial role in the protein glycosylation process, the molecular mechanism of heteromeric cis-PTases remains poorly understood due to lack of structural-functional studies on these enzymes, in contrast to homodimeric cis-PTases which have been extensively studied. Therefore, in this dissertation, I illustrate the first crystal structure of a heteromeric, human cis-PTase NgBR/DHDDS complex solved at 2.3 Å. The structure revealed novel features that were not previously observed in homodimeric enzymes, including a new dimeric interface formed by a unique C-terminus in DHDDS and a novel N-terminal segment in DHDDS serving as a membrane sensor for lipid activation. In addition, the structure elucidated the molecular details associated with substrate binding, catalysis, and disease-causing mutations. Finally, the structure provided novel insights into the mechanism of product chain elongation, an interesting yet one of the most enigmatic topics on prenyl chain elongating enzymes. In summary, the crystal structure advances our understanding of the molecular mechanism of heteromeric cis-PTase enzymes

Structural elucidation of the cis -prenyltransferase NgBR/DHDDS complex reveals insights in regulation of protein glycosylation

Cis- prenyltransferase ( cis- PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis -PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme’s active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis -PTase activity. Comparison of NgBR/DHDDS with homodimeric cis -PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme’s active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis -PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy, and movement disorder. We evaluated a large cohort of patients (n=25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor, and ataxia. Later in the disease course they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration, and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibers and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders

The Missing Electrostatic Interactions Between DNA Substrate and Sulfolobus Solfataricus DNA Photolyase: What Is the Role of Charged Amino Acids in Thermophilic DNA Binding Proteins?

DNA photolyase can be used to study how a protein with its required cofactor has adapted over a large temperature range. The enzymatic activity and thermodynamics of substrate binding for protein from Sulfolobus solfataricus were directly compared to protein from Escherichia coli. Turnover numbers and catalytic activity were virtually identical, but organic cosolvents may be necessary to maintain activity of the thermophilic protein at higher temperatures. UV-damaged DNA binding to the thermophilic protein is less favorable by ∼2 kJ/mol. The enthalpy of binding is ∼10 kJ/mol less exothermic for the thermophile, but the amount and type of surface area buried upon DNA binding appears to be somewhat similar. The most important finding was observed when ionic strength studies were used to separate binding interactions into electrostatic and nonelectrostatic contributions; DNA binding to the thermophilic protein appears to lack the electrostatic contributions observed with the mesophilic protein

Data related to article "De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus"

Clinical data of a patient included at study at titl