Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

Abstract

We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH 4 ) in the lipidproducing fungus Mortierella alpina. The BH 4 cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H 2 -NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H 2 -NTP to 6-pyruvoyltetrahydropterin (PPH 4 ), and sepiapterin reductase (SR) for the conversion of PPH 4 to BH 4 , were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH 4 in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH 4 and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH 4 biosynthesis pathway in a fungus.

Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

“…The fungal BH 4 synthesis pathway has been characterized at the molecular level only in M. alpina (22), and BH 4 acts as a cofactor in nitric oxide synthesis only in the fungi P. blakesleeanus and Neurospora crassa (70,71). Because most fungi do not synthesize BH 4 and lack BH 4 -dependent monooxygenases, the function of BH 4 in fungi is unclear.…”

“…Genes encoding PCD and DHPR, which are required for the regeneration of BH 4 , an essential component of the phenylalanine-hydroxylating system (6), were also found in the M. alpina genome. The BH 4 de novo synthesis pathway in M. alpina was first purified and characterized in our laboratory (22), but the function of BH 4 in fungi is still not well understood. The presence of the phenylalanine-hydroxylating system in M. alpina suggests that fungi can make use of the system in their phenylalanine degradation strategy.…”

Nitric oxide: an effective weapon of the plant or the pathogen?

“…Unlike plants, which do not synthesize tetrahydrobiopterin (BH 4 ), but convert its first intermediate (i.e. dihydroneopterin triphosphate, H 2 ‐NTP) into folate (Stanger, ; Wang et al ., ), both the ascomycete N. crassa and zygomycete P. blakesleeanus are equipped with BH 4 , a typical mammalian NOS cofactor essential for NO synthesis (Maier and Ninnemann, ).…”

How complexity originates: The evolution of animal eyes

“…Pterin synthesis involves the conversion of the purine GTP to H 4 biopterin through the enzymatic actions of GTP cyclohydrolase 1 (GCH1), 6-pyruvoyl H 4 pterin synthase (PTS), and sepiapterin reductase (SPR) (100,105). All three of these enzymes originated prior to the appearance of metazoans: GCH1, PTS, and SPR contribute to the synthesis of H 4 biopterin in the fungus Mortierella alpine (110) and phylogenetic evidence suggests that homologs of these genes may be present in bacteria (78). The components of the ommochrome synthesis pathway include tryptophan 2,3-dioxygenase (TDO2), kynurenine formamidase (KF), and kynurenine 3-monooxygenase (KMO) (87, 88, 100), all of which are deeply conserved among metazoans (78) and beyond (e.g.…”