Biopterin is an essential cofactor in mammalian nitric oxide synthase and aromatic hydroxylation reactions, but is not commonly found in bacteria. Glycosylated biopterin analogs are synthesized by a number of photosynthetic bacteria and may be involved in photoreception or UV protection (Wachi et al.
; Yamazawa et al.
). Tetrahydrobiopterin (BH4
) is biosynthesized in three steps from GTP by the successive actions of GTP cyclohydrolase I (GCH I), 6-pyruvoyltetrahydrobiopterin synthase (PTPS) and sepiapterin reductase (SR). PTPS is a zinc-containing protein that catalyzes two successive Amadori rearrangements to convert 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin.
The hexameric rat PTPS assumes a ‘tunnel’ fold (T-fold; Nar et al.
; PDB code 1b66
), which has also been reported for GTP cyclohydrolase I (GCH I; Nar et al.
; PDB code 1gtp
), dihydroneopterin aldolase/epimerase (DHN aldolase; Sanders et al.
; PDB code 1rrw
) and urate oxidase (Retailleau et al.
; PDB code 1r51
). The T-fold of PTPS contains a dimer of trimers that form a stacked toroid. The active site of PTPS lies at the interface of three subunits (A
′ and B
; Ploom et al.
; see gray residues in Fig. 1). Three histidine imidazole side chains from subunit A
(His23, His48 and His50) and the hydroxyl group at C2′ of the substrate coordinate an essential divalent zinc metal ion in the active site. An Asp88-His89 duo from subunit B
activates Cys42 of subunit A
to initiate the catalytic cycle (see Fig. 1). The endocyclic nitrogen at N3 and the exocyclic amino group at C2 hydrogen bond to the side-chain carboxylate of Glu107, while the backbone amide of the preceding residue (Thr106) hydrogen bonds to the pyrimidine carbonyl O atom (at C3). Additionally, a second glutamic acid side chain from subunit A
(Glu133) forms a hydrogen bond to the C1′ hydroxyl of the substrate (Fig. 1). While subunit A
′ is not directly involved in catalysis, it lines one side of the active site and the backbone carbonyl of Met70 forms a hydrogen bond to the exocyclic amino group, presumably stabilizing the bound substrate.
Figure 1 Summary of interactions between Rattus rattus PTPS and its substrate, 7,8-dihydroneopterin triphosphate (PDB code 1b66). The active site is composed of residues from three adjacent subunits (A, A′ and B). For each residue, the contributing (more ...)
The genome sequence of Streptomyces coelicolor
(Bentley et al.
) revealed the presence of a PTPS homolog (SCO 6650), which is nestled in a cluster of genes that appears to be conserved in all strains of actinomyces sequenced to date. In addition to PTPS, this cluster also contains a GTP cyclohydrolase II homolog, SCO 6655, which we have shown to catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H
)-pyrimidinone 5′-phosphate (FAPy; Spoonamore et al.
). A biological imperative for the co-localization of these genes is not clear.
As part of a program to assign the biological function of this cluster, SCO 6650 was cloned, overexpressed in Escherichia coli and purified as either His6-tagged or native protein. Both the native and His6-tagged proteins crystallized readily and the structure of the selenomethionine-substituted His6-tagged protein was solved to 1.5 Å resolution. The model reveals that SCO 6650 lacks nearly all the catalytically essential residues but retains the residues required for binding a pterin-like substrate.