moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans

Abstract

Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/ moc-6 , the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/ moc-6 and with these alleles genetically demonstrate that K10D2.7/ moc-6 is necessary for endogenous Moco synthesis in C. elegans.

Obtaining the necessary molybdenum cofactor for sulfite oxidase activity in the nematode Caenorhabditis elegans surprisingly involves a dietary source

“…moc-6 encodes another component of the Moco-biosynthetic machinery and is necessary for C. elegans Moco synthesis ( Fig. 1 ) ( 12 ). Supporting our findings with moc-4; cdo-1 animals, we were unable to detect SUOX-1 activity in crude extracts from moc-6; cdo-1 double mutant animals fed Moco- E. coli ( Fig.…”

“…We then sought to test the role of endogenous Moco synthesis in supporting the growth and development of suox-1(gk738847) mutant C. elegans. To test this, we attempted to engineer double mutant strains of C. elegans by combining the suox-1(gk738847) allele with various mutations in C. elegans Moco-biosynthetic enzymes ( moc-1(ok366) , moc-4(ok2571) , moc-5(mg589) , and moc-6(rae296) ) ( 12 , 14 ) . In our attempts to construct these double mutants, we were unable to isolate viable double mutant strains.…”

“…To expand our understanding of Moco homeostasis, we produced C. elegans extracts from animals lacking either endogenous Moco synthesis or dietary Moco. These extracts were used to quantify Moco content and SUOX-1 activity, providing biochemical clarity to our genetic system ( 12 , 13 , 14 ). We found that C. elegans mutants lacking endogenous Moco biosynthesis displayed reduced Moco content and SUOX-1 activity.…”

“…All samples were cultured on either BW25113 (WT, Moco+) or JW0764-2 ( ΔmoaA753::kan, Moco-) E. coli for the entire experiment. However, if moc- mutant C. elegans are cultured on ΔmoaA (Moco-) E. coli from early larval stages, they undergo larval arrest and death ( 12 , 14 ). To allow for biochemical analyses of moc-4 and moc-6 single mutant animals cultured on ΔmoaA (Moco-) E. coli , these mutant strains were initially cultured on Moco+ E. coli until the L4 stage of development (48 h post L1 synchronization).…”

“…Most Moco-requiring cells are believed to synthesize Moco de novo. However, recent advances in the nematode Caenorhabditis elegans have demonstrated that Moco can move not only between cells and tissues but between disparate organisms ( 12 , 13 , 14 ). Unlike other animals studied, C. elegans with null mutations in genes encoding the Moco-biosynthetic enzymes are viable and fertile.…”