Regulation of Tetrahydrobiopterin (BH4) Synthesis in the Nematode C. elegans

Abstract

5,6,7,8‐tetrahydrobiopterin (BH4) is a cofactor required by the aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases, and alkylglycerol monooxygenase (Werner et al., 2011, Biochem. J. 438: 397). Among the AAAHs are the rate‐limiting synthetic enzymes for serotonin and dopamine synthesis, and for conversion of phenylalanine (Phe) to tyrosine. In humans, the activity of phenylalanine hydroxylase (PAH) is essential for Phe catabolism; dysfunction of PAH, either from mutations in the PAH gene or in genes required for BH4 synthesis, results in phenylketonuria. Regulation of BH4 levels is important for many other physiological functions. The first and rate‐limiting step in BH4 de novo synthesis is performed by GTP Cyclohydrolase I (GTPCH1). Like in many biosynthesis pathways, this first enzyme is regulated by ‘end product feedback inhibition’ whereby BH4 inhibits GTPCH1 activity. In mammals, BH4 does not bind to GTPCH1 alone, but requires an additional protein called GTPCH1 feedback regulatory protein (GFRP) to inhibit GTPCH1. Crystal structures of rat GTPCH1 and GFRP with the BH4 analog BH2 show that the biopterin binds at the interface between the proteins (Maita et al., 2004, J Biol Chem 279: 51534). Phe increases BH4 production via GTPCH1 stimulation, also in a GFRP‐dependent fashion, similarly binding at the interface between GTPCH1 and GFRP. We have recently described the function and expression of genes required for BH4 synthesis and regeneration in the nematode C. elegans (Loer et al., 2015, Genetics 200: 237). Here we present work on the function of the gene gfrp‐1, which encodes a ortholog of mammalian GFRP, in the regulation of BH4 synthesis in C. elegans. Sequence analysis and structural predictions suggest the worm GFRP 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). Using a reduction‐of‐function mutant in the worm GTPCH1 gene [cat‐4(e3015) – S148N], we find that DAHP reduces BH4 production, as assessed by a significant reduction in serotonin immunoreactivity in whole worms. Treatment with Phe significantly increases serotonin levels in cat‐4(e3015) worms. We are currently assessing the effects of mutations in gfrp‐1 on serotonin levels (hence BH4 levels) in single mutants and cat‐4; gfrp‐1 double mutants, and whether the effects of DAHP and Phe on BH4 synthesis are GFRP‐dependent.Support or Funding InformationSupported by a Univ of San Diego Faculty Research Grant and an endowment from the Fletcher Jones Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.