Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.

Abstract

The crystal structure of rat liver 6‐pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R‐factor of 20.4% at 2.3 A resolution. 6‐Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6‐pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6‐pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four‐stranded, antiparallel beta‐sheet with a 25 residue, helix‐containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta‐sheet. Three 6‐pyruvoyl tetrahydropterin synthase monomers form an unusual 12‐stranded antiparallel beta‐barrel by tight association between the N‐ and C‐terminal beta‐strands of two adjacent subunits. The barrel encloses a highly basic pore of 6‐12 A diameter. Two trimers associate in a head‐to‐head fashion to form the active enzyme complex. The substrate‐binding site is located close to the trimer‐trimer interface and comprises residues from three monomers: A, A’ and B. A metal‐binding site in the substrate‐binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6‐pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.

Active site topology and reaction mechanism of GTP cyclohydrolase I.

“…This domain has a central fourstranded antiparallel 13-sheet that is flanked on both sides by a-helices. The folding topology of this domain is identical to that of a subunit of the second enzyme in tetrahydrobiopterin biosynthesis pathway, 6-pyruvoyltetrahydropterin synthase (20).…”

Zinc plays a key role in human and bacterial GTP cyclohydrolase I

“…The structures of Escherichia coli GTP cyclohydrolase I (eGTP-CH-I) (7), rat liver 6-pyruvoyl tetrahydropterin synthase (8,9), and mouse sepiapterin reductase (10) have been determined by x-ray crystallography; eGTP-CH-I is a toroid-shaped, D5-symmetric homodecamer (11). The 10 equivalent active sites are located at the periphery of the toroid, and each catalytic site is located at the interface of three adjacent subunits.…”

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

“…ture they produce distinctly different major products. The active sites of both proteins are composed of a constellation of residues that are contributed by adjacent subunits in the biological assembly (34). The substrate in each active site is bound to a conserved zinc divalent cation via the C1Ј and C2Ј hydroxyl groups and the proteins retain similar binding interactions with the substrate (35).…”