Background on Pteridines

Pteridines are compounds containing the bicyclic pteridine ring system, whose atoms are numbered as shown in Figure 1. There are two classes of naturally occurring pteridines – pterins and lumazines. Pterins have an amino group in the 2-position and an oxo group in the 4-position, and are derivatives of the parent compound pterin (Fig. 1). Most natural pteridines are pterins, and have substituents at positions 6 or 7. Pterins can be reduced to the corresponding dihydro and tetrahydro forms (Fig. 1), and this is the basis for their activity as redox cofactors. The reduced forms often predominate in vivo. Lumazines have oxo groups at both 2- and 4-positions, and are derived from pterins by deamination. The simplest is lumazine (Fig. 1).
 
 

The substituents at the 6- or 7-positions range from a hydroxyl group through one, two, and three-carbon side chains, which in turn may be coupled to other groups. In some cases, the side chains have one or two asymmetric centers, giving rise to pairs of diastereomers. Figure 2 shows the substituents of several naturally occuring pterins.
 
 

Natural pteridines are easily oxidized and light-sensitive. The di- or tetrahydro forms are more unstable and photo-labile than the fully oxidized (aromatic) forms (Rembold & Gyure, 1972; Pfleiderer, 1984), especially at neutral and alkaline pH. For example, 6-substituted pteridines such as dihydroneopterin (DHN) and tetrahydrobiopterin oxidize readily and lose all or part of their side chains (Rembold & Gyure, 1972; Pfleiderer, 1984). Pteridines have characteristic UV absorption spectra and their aromatic forms are strongly fluorescent; these features are useful in identification and measurement (Rembold & Gyure, 1972; Pfleiderer, 1984). Depending on the side chain, absorption bands above 350 nm can occur; for example, sepiapterin has one at ~420 nm and is yellow. Pterins are amphoteric, with a basic pKa due to protonation at N-1 and an acidic pKa due to the 4-oxo group (Pfleiderer, 1984). Pteridines are generally poorly water-soluble when uncharged and more soluble when ionized (Pfleiderer, 1984; Nixon, 1984); some (e.g., isoxanthopterin) are acid-insoluble (Rembold & Gyure, 1972).

Pteridines in Plant Folate Synthesis

The folate synthesis pathway in plants has the same steps as in microorganisms, but is split between three subcellular compartments as shown in Figure 3 (Green et al., 1996; Hanson & Gregory, 2002). The branch involving pterins (blue) is mainly cytosolic. GTP is converted to DHN triphosphate (DHNTP) (Basset et al., 2002), followed by a two-step dephosphorylation to give DHN, and then by aldol cleavage of the trihydroxypropyl side chain to yield 6-hydroxymethyldihydropterin (HMDHP). The DHN aldolase that mediates this cleavage also catalyzes epimerization at the second carbon of the side chain, producing dihydromonapterin (DHM), which can also undergo the cleavage reaction (Goyer et al., 2004). The subsequent steps of folate synthesis, starting with pyrophosphorylation of HMDHP, are all mitochondrial (Rébeillé & Douce, 1999; Hanson & Gregory, 2002).
 
 

Enzymes for all specific steps in the pterin branch of folate synthesis have been cloned from bacteria and plants, except for one (in red, Fig. 3). This uncloned enzyme, DHN triphosphate pyrophosphatase (DTPase), is of special interest as it may mediate a reaction that commits pterins to folate synthesis (Suzuki & Brown, 1974; Lee et al., 1999).

USDA-NRI Grant # 2008-35318-04589

Pterin-linked aromatic hydroxylases from pine and moss: Biochemical and reverse-genetic characterization of a new class of plant enzymes

Pterin-linked aromatic amino acid hydroxylases (AAHs) have been considered specific to animals and bacteria. They convert aromatic amino acids to ring-hydroxylated derivatives e.g., phenylalanine (Phe) ® tyrosine (Tyr) using a tetrahydropterin as electron donor (Thony et al. 2000). The resulting oxidized pterin is recycled to the tetrahydro level by a pterin carbinolamine dehydratase (PCD) and a reductase (Figure 1). Animal and bacterial Phe hydroxylases initiate Phe catabolism via the homogentisate pathway (Arias-Barrau et al. 2004).

Figure 1. The cofactor regeneration cycle. The requirement for a tetrahydropterin (H4-pterin) cofactor for phenylalanine hydroxylase (AAH) and the cofactor regeneration cycle involving pterin-4a-carbinolamine dehydratase (PCD) and quinonoid dihydropterin (q-H2-pterin) reductase (q-DHPR).

Surprisingly, genome analysis revealed AAH and PCD genes in conifers and mosses. Phylogenomic and functional analysis of the plant PCDs established that plants have functional PCDs (Naponelli et al. 2008). We are currently investigating the biochemical and functional characterization of the plant AAHs.

References

Arias-Barrau E, Olivera ER, Luengo JM, Fernandez C, Galan B, Garcia JL, Diaz E, Minambres B (2004) The homogentisate pathway: a central catabolic pathway involved in the degradation of L-Phenylalanine, L-Tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida. J Bacteriol 186: 5062-5077.

Basset G, Quinlivan EP, Ziemak MJ, Diaz de la Garza R, Fischer M, Schiffmann S, Bacher A, Gregory JF 3rd, Hanson AD (2002) Folate synthesis in plants: the first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proc Natl Acad Sci USA 99: 12489-12494

Green JC, Nichols BP, Matthews RG (1996) Folate biosynthesis, reduction, and polyglutamylation. In: Escherichia coli and Salmonella - Cellular and Molecular Biology, Vol. 1. Neidhardt FC et al., eds. Washington DC, ASM Press, pp. 665-673

Goyer A, Illarionova V, Roje S, Fischer M, Bacher A, Hanson AD (2003) Folate biosynthesis in higher plants: cDNA cloning, heterologous expression, and characterization of multiple dihydroneopterin aldolases. Plant Physiol 135: 1-9

Hanson AD, Gregory JF 3rd (2002) Synthesis and turnover of folates in plants. Curr Opin Plant Biol 5: 244-249

Klaus SM, Wegkamp A, Sybesma W, Hugenholtz J, Gregory JF 3rd, Hanson AD (2005). A nudix enzyme removes pyrophosphate from dihydroneopterin triphosphate in the folate synthesis pathway of bacteria and plants. J Biol Chem. 280(7):5274-80

Lee SW, Lee HW, Chung HJ, Kim YA, Kim YJ, Hahn Y, Chung JH, Park YS (1999) Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803. FEMS Microbiol Lett 176: 169-176

Naponelli V, Noiriel A, Ziemak MJ, Beverley SM, Lye LF, Plume AM, Botella JR, Loizeau K, Ravanel S, Rébeillé F, de Crécy-Lagard V, Hanson AD (2008) Phylogenomic and Functional Analysis of Pterin-4a-Carbinolamine Dehydratase Family (COG2154) Proteins in Plants and Microorganisms. Plant Physiol 146: 1515-27.