A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

ACAD10 and ACAD11 allow entry of 4-hydroxy fatty acids into ß-oxidation.

Hydroxylated fatty acids are important intermediates in lipid metabolism and signaling. Surprisingly, the metabolism of 4-hydroxy fatty acids remains largely unexplored. We found that both ACAD10 and ACAD11 unite two enzymatic activities to introduce these metabolites into mitochondrial and peroxisomal ß-oxidation, respectively. First, they phosphorylate 4-hydroxyacyl-CoAs via a kinase domain, followed by an elimination of the phosphate to form enoyl-CoAs catalyzed by an acyl-CoA dehydrogenase (ACAD) domain. Studies in knockout cell lines revealed that ACAD10 preferentially metabolizes shorter chain 4-hydroxy fatty acids than ACAD11 (i.e. 6 carbons versus 10 carbons). Yet, recombinant proteins showed comparable activity on the corresponding 4-hydroxyacyl-CoAs. This suggests that the localization of ACAD10 and ACAD11 to mitochondria and peroxisomes, respectively, might influence their physiological substrate spectrum. Interestingly, we observed that ACAD10 is cleaved internally during its maturation generating a C-terminal part consisting of the ACAD domain, and an N-terminal part comprising the kinase domain and a haloacid dehalogenase (HAD) domain. HAD domains often exhibit phosphatase activity, but negligible activity was observed in the case of ACAD10. Yet, inactivation of a presumptive key residue in this domain significantly increased the kinase activity, suggesting that this domain might have acquired a regulatory function to prevent accumulation of the phospho-hydroxyacyl-CoA intermediate. Taken together, our work reveals that 4-hydroxy fatty acids enter mitochondrial and peroxisomal fatty acid ß-oxidation via two enzymes with an overlapping substrate repertoire.

A novel gluconeogenic route enables efficient use of erythritol in zoonotic Brucella.

Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus Brucella. While most B. abortus, B. melitensis, and B. suis biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses in vitro and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While Brucella sugar and erythritol catabolic pathways are known, growth on 3-4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of B. suis biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Brucella Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in B. abortus and B. melitensis, GlpX is disabled in B. suis biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we re-examined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp-Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in Brucella. We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals.

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene.

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

Clinical, Neuroimaging, and Metabolic Footprint of the Neurodevelopmental Disorder Caused by Monoallelic HK1 Variants.

Background and Objectives: Hexokinase 1 (encoded by HK1) catalyzes the first step of glycolysis, the adenosine triphosphate-dependent phosphorylation of glucose to glucose-6-phosphate. Monoallelic HK1 variants causing a neurodevelopmental disorder (NDD) have been reported in 12 individuals. Methods: We investigated clinical phenotypes, brain MRIs, and the CSF of 15 previously unpublished individuals with monoallelic HK1 variants and an NDD phenotype. Results: All individuals had recurrent variants likely causing gain-of-function, representing mutational hot spots. Eight individuals (c.1370C>T) had a developmental and epileptic encephalopathy with infantile onset and virtually no development. Of the other 7 individuals (n = 6: c.1334C>T; n = 1: c.1240G>A), 3 adults showed a biphasic course of disease with a mild static encephalopathy since early childhood and an unanticipated progressive deterioration with, e.g., movement disorder, psychiatric disease, and stroke-like episodes, epilepsy, starting in adulthood. Individuals who clinically presented in the first months of life had (near)-normal initial neuroimaging and severe cerebral atrophy during follow-up. In older children and adults, we noted progressive involvement of basal ganglia including Leigh-like MRI patterns and cerebellar atrophy, with remarkable intraindividual variability. The CSF glucose and the CSF/blood glucose ratio were below the 5th percentile of normal in almost all CSF samples, while blood glucose was unremarkable. This biomarker profile resembles glucose transporter type 1 deficiency syndrome; however, in HK1-related NDD, CSF lactate was significantly increased in all patients resulting in a substantially different biomarker profile. Discussion: Genotype-phenotype correlations appear to exist for HK1 variants and can aid in counseling. A CSF biomarker profile with low glucose, low CSF/blood glucose, and high CSF lactate may point toward monoallelic HK1 variants causing an NDD. This can help in variant interpretation and may aid in understanding the pathomechanism. We hypothesize that progressive intoxication and/or ongoing energy deficiency lead to the clinical phenotypes and progressive neuroimaging findings.