-Prenyltransferases (-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, -PT is the first enzyme committed to the synthesis of dolichyl phosphate, an obligate lipid carrier in protein glycosylation reactions. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase, generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently, we discovered that unlike undecaprenyl diphosphate synthase, mammalian-PT is a heteromer consisting of NgBR (Nus1) and hCIT (dehydrodolichol diphosphate synthase) subunits, and this composition has been confirmed in plants and fungal -PTs. Here, we establish the first purification system for heteromeric-PT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical RG sequence in the C-terminal tail of NgBR that is conserved and essential for enzyme activity across phyla. In summary, our findings show that eukaryotic -PT is composed of the NgBR and hCIT subunits. The strong conservation of the RG motif among NgBR orthologs indicates that this subunit is critical for the synthesis of polyprenol diphosphates and cellular function.
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.
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.
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 the first 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 3 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.
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.
2 Summarycis-Prenyltransferases (cisPTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, cisPT is the first enzyme committed to the synthesis of dolichyl-phosphate (DolP). DolP is obligate lipid carrier in protein glycosylation reactions in mammals. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase (UPPS) generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently our group discovered that unlike UPPS, mammalian cisPT is a heteromer consisting of NgBR (NUS1) and hCIT (DHDDS) subunits and this composition has been confirmed in plants and fungal cisPTs. Here, we establish the first purification system for heteromeric cisPT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical RxG sequence in the Cterminal tail of NgBR that is conserved and essential for enzyme activity across phyla.
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