Synthesis of the neurotransmitters serotonin and dopamine, conversion of Phe to Tyr, and the breakdown of ether lipids all involve enzymes that require the cofactor 5,6,7,8-tetrahydrobiopterin (BH4) to function. Worms that can't synthesize BH4 are serotonin- and dopamine-deficient, and have leaky, fragile cuticles caused by aberrant lipid metabolism in the hypodermis. The defective cuticle in BH4-deficient worms also alters their susceptibility to bacterial pathogens (Loer et al., 2015, Genetics 200: 237). In humans, regulation of BH4 levels is important for several physiological functions. BH4 is synthesized in four steps starting with GTP; the first step is performed by GTP Cyclohydrolase I (GTPCH1, C. elegans gene
cat-4). Like in many biochemical synthesis pathways, the first enzyme in the pathway is regulated by 'end product feedback inhibition.' This typically occurs by the end product itself binding to another site on the enzyme - allosteric regulation. For BH4, it is more complicated - in mammals, GTPCH1 inhibition by BH4 requires an additional small protein called feedback regulatory protein (GFRP). Crystal structures show that the analog BH2 binds at the interface between pentamers of GTPCH1 & GFRP to inhibit GTPCH1 function, mainly interacting with GTPCH1 (Maita et al., 2004, J Biol Chem 279: 51534). Phe, which stimulates GTPCH1, also binds at the interface between the two proteins, mainly interacting with GFRP. C. elegans encodes a homolog of GFRP (gene
gfrp-1) that at least partly overlaps in expression with the
cat-4 gene (Loer et al., 2015). We are seeking to demonstrate the function of the putative worm GFRP in regulating BH4 synthesis. Sequence analysis and structural predictions of the putative worm GFRP suggest that it will function like the mammalian protein, binding both BH4 and Phe. In mammals, GTPCH1 and GFRP can also bind the selective inhibitor 2,4-Diamino-6-hydroxypyrimidine (DAHP), which is structurally similar to both GTP (substrate) and BH4 (pathway end product). At low concentrations, DAHP apparently inhibits GTPCH1 like BH4 by binding with GFRP; this inhibition is GFRP-dependent. At higher concentrations, DAHP acts as a competitive inhibitor, binding at the GTPCH1 active site like GTP; this inhibition is GFRP-independent (Xie et al., 1998, J Biol Chem 273: 21091). Although DAHP treatment has no obvious effect on wildtype C. elegans, serotonin levels (a proxy for BH4 levels) are reduced by DAHP in worms with a reduction-of-function mutation [
cat-4(
e3015)] that already have less BH4. We plan to test whether this presumed inhibition of worm GTPCH1 is GFRP-dependent by knocking down
gfrp-1 gene function in DAHP-treated
cat-4(
e3015) worms.