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.

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Four naturally occurring mutants with single amino acid alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were overexpressed and characterized in vitro. The corresponding DNA mutations were found in patients with hyperphenylalaninemia and monoamine neurotransmitter insufficiency due to lack of the tetrahydrobiopterin biosynthetic enzyme PTPS. To predict the structure of the mutant enzymes, computer modeling was performed based on the solved three-dimensional structure of the homohexameric rat enzyme. One mutant (delta V57) is incorrectly folded and thus unstable in vitro and in vivo, while a second mutant (P87L) has substantial activity but enhanced sensitivity to local unfolding. Two other mutants, R16C and R25Q, form stable homomultimers and exhibit significant activity in vitro but no activity in COS-1 cells. In vivo 32P labeling showed that wild-type PTPS, P87L, and R25Q are phosphorylated, while R16C is not modified. This strongly suggests that the serine 19 within the consensus sequence for various kinases, RXXS, is the site of modification. Our results demonstrate that PTPS undergoes protein phosphorylation and requires additional, not yet identified post-translational modification(s) for its in vivo function.

Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.