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Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy.

ABSTRACT: Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B6 vitamer metabolism or by inactivation of PLP, which can occur when compounds accumulate as a result of inborn errors of other pathways or when small molecules are ingested. Whole-exome sequencing of two children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial), PROSC, which encodes a PLP-binding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified four additional children with biallelic PROSC mutations. Pre-treatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant lacking the PROSC homolog (?YggS) is pyridoxine sensitive; complementation with human PROSC restored growth whereas hPROSC encoding p.Leu175Pro, p.Arg241Gln, and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells, which is how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Although the mechanism involved is not fully understood, our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions.

SUBMITTER: Darin N

PROVIDER: S-EPMC5142116 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy.

Darin Niklas N Reid Emma E Prunetti Laurence L Samuelsson Lena L Husain Ralf A RA Wilson Matthew M El Yacoubi Basma B Footitt Emma E Chong W K WK Wilson Louise C LC Prunty Helen H Pope Simon S Heales Simon S Lascelles Karine K Champion Mike M Wassmer Evangeline E Veggiotti Pierangelo P de Crécy-Lagard Valérie V Mills Philippa B PB Clayton Peter T PT

American journal of human genetics 20161201 6

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B6 vitamer metabolism or by inactivation of PLP, which can occur when compounds accumula ...[more]

PMID: 27912044

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Project description:BACKGROUND: The pyridoxal-5'-phosphate (PLP)-dependent or vitamin B6-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B6 enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase), in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP. RESULTS: We screened the PLP adduct of wild-type RNase and two variant RNases (K7R and K41R) for catalytic effects toward L- and D-amino acids. RNase(K41R)-PLP, in which the cofactor is bound through an imine linkage to Lys7, qualifies for a model proto-B6 enzyme by the following criteria: (1) covalent linkage of PLP (internal aldimine); (2) catalytic activity toward amino acids that depends on formation of an imine linkage with the substrate (external aldimine); (3) adjoining binding sites for the cofactor and amino acid moiety that facilitate the transimination reaction of the internal to the external aldimine and stabilize the resulting noncovalent complex of the coenzyme-substrate adduct with the protein; (4) reaction specificity, the only detectable reactions being racemization of diverse amino acids and beta-decarboxylation of L-aspartate; (5) acceleration factors for racemization and beta-decarboxylation of >103 over and above that of PLP alone; (6) ribonuclease activity that is 103-fold lower than that of wild-type RNase, attenuation of a pre-existing biological activity being indispensable for the further evolution as a PLP-dependent protoenzyme. CONCLUSION: A single amino acid substitution (Lys41Arg) and covalent binding of PLP to active-site Lys7 suffice to turn pancreatic ribonuclease A into a protein catalyst that complies with all plausible criteria for a proto-B6 enzyme. The study thus retraces in a model system what may be considered the committed step in the molecular evolution of a potential ancestor of a B6 enzyme family.

Dataset Information

Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy.

Publications

Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B<sub>6</sub>-Dependent Epilepsy.

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