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

Abstract

Molybdenum cofactor (Moco) is a prosthetic group necessary for the activity of four unique enzymes, including the essential sulfite oxidase (SUOX-1). Moco is required for life; humans with inactivating mutations in the genes encoding Moco-biosynthetic enzymes display Moco deficiency, a rare and lethal inborn error of metabolism. Despite its importance to human health, little is known about how Moco moves among and between cells, tissues, and organisms. The prevailing view is that cells that require Moco must synthesize Moco de novo. Although, the nematode Caenorhabditis elegans appears to be an exception to this rule and has emerged as a valuable system for understanding fundamental Moco biology. C. elegans has the seemingly unique capacity to both synthesize its own Moco as well as acquire Moco from its microbial diet. However, the relative contribution of Moco from the diet or endogenous synthesis has not been rigorously evaluated or quantified biochemically. We genetically removed dietary or endogenous Moco sources in C. elegans and biochemically determined their impact on animal Moco content and SUOX-1 activity. We demonstrate that dietary Moco deficiency dramatically reduces both animal Moco content and SUOX-1 activity. Furthermore, these biochemical deficiencies have physiological consequences; we show that dietary Moco deficiency alone causes sensitivity to sulfite, the toxic substrate of SUOX-1. Altogether, this work establishes the biochemical consequences of depleting dietary Moco or endogenous Moco synthesis in C. elegans and quantifies the surprising contribution of the diet to maintaining Moco homeostasis in C. elegans.

Keywords: Caenorhabditis elegans; diet; genetics; inborn error of metabolism; molybdenum.

Figure 1

Intersection of Moco biosynthesis and sulfur amino acid metabolism in C. elegans. The C. elegans Moco biosynthetic and sulfur amino acid metabolism (simplified) pathways are displayed (enzymes, red). Structures of the following Moco-biosynthetic intermediates are displayed: guanosine triphosphate (GTP), cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), molybdopterin adenosine monophosphate (MPT-AMP), and molybdenum cofactor (Moco). Moco, molybdenum cofactor.

Figure 2

Quantifying SUOX-1 activity in crude extracts from C. elegans.A, simplified reaction mechanism for SUOX-1 by which sulfite (SO₃^2-) is oxidized to sulfate (SO₄^2-). Sulfite-dependent SUOX-1 activity is detected via the concomitant reduction of cytochrome c. B, SUOX-1 activity was detected in crude extracts from WT C. elegans fed WT (Moco+) E. coli (black) or moc-4(ok2571); cdo-1(mg622) mutant C. elegans fed ΔmoaA (Moco-) E. coli (red). Units of SUOX-1 activity per milligram of protein are displayed. C, SUOX-1 activity is displayed for WT and mutant C. elegans fed either WT (Moco+) or ΔmoaA (Moco-) E. coli. All biological replicates, the sample size, mean, and SD are displayed for each condition. N/D indicates no SUOX-1 activity was detected in any sample. ∗∗∗∗p < 0.0001, ordinary one-way ANOVA with Dunnet’s post hoc analysis. Note, the WT data in Figure 2C are also displayed in Figure 4, Figure 5, Figure 6A, to allow for appropriate graphical comparisons of data. Moco, molybdenum cofactor.

Figure 3

Quantifying Moco content in crude extracts from C. elegans.A, a schematic of the conversion of Moco to dephospho-FormA (_dpFormA) in the presence of an acidic iodine environment and treatment with alkaline phosphatase. B, HPLC measurements of Moco-derived _dpFormA from crude extracts of WT C. elegans fed WT (Moco+) E. coli (black) or moc-4(ok2571); cdo-1(mg622) mutant C. elegans fed ΔmoaA (Moco-) E. coli (blue). The _dpFormA peak is indicated (black arrow). C, _dpFormA content is displayed for WT, and mutant C. elegans fed either WT (Moco+) or ΔmoaA (Moco-) E. coli. All biological replicates, the sample size, mean, and SD are displayed for each condition. N/D indicates no _dpFormA was detected in any sample. ∗∗∗∗p < 0.0001, ordinary one-way ANOVA with Dunnet’s post hoc analysis. Note, the WT data in Figure 3C are also displayed in Figures 4B and 5B to allow for appropriate graphical comparisons of data. Moco, molybdenum cofactor.

Figure 4

Dietary Moco is necessary to promote SUOX-1 activity and Moco accumulation.A, SUOX-1 activity and (B) _dpFormA content are displayed for WT C. elegans fed either WT (Moco+) or ΔmoaA (Moco-) E. coli. All biological replicates, the sample size, mean, and SD are displayed for each condition. ∗∗∗∗p < 0.0001, Welch’s t test. Moco, molybdenum cofactor.

Figure 5

Endogenous Moco synthesis is necessary to promote SUOX-1 activity and Moco accumulation.A, SUOX-1 activity and (B) _dpFormA content are displayed for WT, moc-4, and moc-6 mutant C. elegans fed WT (Moco+) E. coli. All biological replicates, the sample size, mean, and SD are displayed for each condition. ∗p < 0.05 and ∗∗∗∗p < 0.0001, ordinary one-way ANOVA with Dunnet’s post hoc analysis. Moco, molybdenum cofactor.

Figure 6

Dietary Moco and endogenous Moco synthesis are nonredundantly required for sulfite tolerance.A, SUOX-1 activity is displayed for WT and suox-1(gk738847) mutant C. elegans fed WT (Moco+) E. coli. All biological replicates, the sample size, mean, and SD are displayed for each condition. ∗∗∗∗p < 0.0001, Welch’s t test. B and C, WT and mutant C. elegans were synchronized at L1 stage and cultured on WT (Moco+) or ΔmoaA mutant (Moco-) E. coli supplemented with various concentrations of sulfite (0, 0.0001, 0.001, 0.0025, 0.005, 0.01, and 0.02M). Animal length was measured after 72 h of growth at 20 °C. Data points represent the average of 3 (suox-1, Moco+ (purple) and moc-4, Moco+ (blue)) or 4 (WT, Moco+ (black) and WT, Moco- (red)) biological replicates. For each biological replicate, 15 or more individual C. elegans animals were imaged and measured at each sulfite concentration. Data points indicate mean and error bars display SD. IC₅₀ was calculated by nonlinear regression analyses and shading indicates the 95% confidence interval. IC₅₀ and R-squared values are displayed. Note that the data displayed for WT C. elegans fed Moco+ E. coli in Figure 6B are identical to the same control displayed in Figure 6C. D, Representative individuals are displayed at the critical 0.005M supplemental sulfite concentration. The scale bar represents 250 μm. Yellow arrowhead indicates embryos in the uterus of the gravid adult animal. Moco, molybdenum cofactor.

Figure 7

Dietary Moco is essential for C. elegans development when suox-1 activity is compromised.A, suox-1(gk738847) mutant C. elegans were synchronized at the L1 stage and cultured on mixtures of WT (Moco+) and ΔmoaA mutant (Moco-) E. coli. Animal length was measured after 72 h of growth at 20 °C. Box plots display the median, upper, and lower quartiles, while whiskers indicate minimum and maximum data points. Sample size is 15 individuals per experiment. ∗∗∗∗p < 0.0001, ordinary one-way ANOVA with Tukey’s post hoc analysis. B, representative individuals from Figure 7A are displayed. The scale bar represents 250 μm. Yellow arrowheads indicate embryos in the uterus of gravid adult animals. Moco, molybdenum cofactor.

Figure 8

C. elegans Moco synthesis is essential when suox-1 activity is compromised.A, suox-1(gk738847), moc-4(+)/moc-4(ok2571); suox-1(gk738847), and moc-4(ok2571); suox-1(gk738847) mutant C. elegans were synchronized at the L1 stage and cultured on WT (Moco+) E. coli. Animal length was measured after 72 h of growth at 20 °C. Box plots display the median, upper, and lower quartiles, while whiskers indicate minimum and maximum data points. Sample size is 15 individuals per experiment. ∗∗∗∗p < 0.0001, ordinary one-way ANOVA, with Tukey’s post hoc analysis. Note, because moc-4; suox-1 double mutant animals are not viable, they (along with moc-4(+)/moc-4(-); suox-1 animals) were derived from the balanced strain USD1011 (see Experimental procedures) (24). B, representative individuals from Figure 8A are displayed. The scale bar represents 250 μm. Yellow arrowheads indicate embryos in the uterus of gravid adult animals. Moco, molybdenum cofactor.

Figure 9

Moco homeostasis in C. elegans. In healthy control animals, WT C. elegans grown on WT E. coli (Moco+), SUOX-1 function and Moco content are high, leading to normal development and sulfite tolerance. SUOX-1 activity and Moco content are dramatically reduced when animals are fed a Moco- diet or when the endogenous synthesis of Moco is disrupted. These deficiencies result in sensitivity to sulfite (SO₃²⁻). Figure created with BioRender. Moco, molybdenum cofactor.