Günter Auerbach

The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co‐factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 Å in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single‐domain α/β‐fold with a central four‐helix bundle connecting two seven‐stranded parallel β‐sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin‐substrates, which positions the substrate side chain C1′‐carbonyl group near Tyr171 OH and NADP C4′N. The catalytic mechanism of SR appears to consist of a NADPH‐dependent proton transfer from Tyr171 to the substrate C1′ and C2′ carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N‐acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N‐acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.

The Crystal Structures of Zea mays and Arabidopsis 4-Hydroxyphenylpyruvate Dioxygenase

The transformation of 4-hydroxyphenylpyruvate to homogentisate, catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPPD), plays an important role in degrading aromatic amino acids. As the reaction product homogentisate serves as aromatic precursor for prenylquinone synthesis in plants, the enzyme is an interesting target for herbicides. In this study we report the first x-ray structures of the plant HPPDs of Zea mays and Arabidopsis in their substrate-free form at 2.0 Å and 3.0 Å resolution, respectively. Previous biochemical characterizations have demonstrated that eukaryotic enzymes behave as homodimers in contrast to prokaryotic HPPDs, which are homotetramers. Plant and bacterial enzymes share the overall fold but use orthogonal surfaces for oligomerization. In addition, comparison of both structures provides direct evidence that the C-terminal helix gates substrate access to the active site around a nonheme ferrous iron center. In the Z. mays HPPD structure this helix packs into the active site, sequestering it completely from the solvent. In contrast, in the Arabidopsis structure this helix tilted by about 60° into the solvent and leaves the active site fully accessible. By elucidating the structure of plant HPPD enzymes we aim to provide a structural basis for the development of new herbicides.

The catalytic activities of monomeric enzymes show complex pressure dependence

High hydrostatic pressures in the biologically relevant range (⩽ 1,200 bar) are known to cause dissociation of oligomeric enzymes in vitro, whereas protein denaturation requires pressures far beyond this range. Pressure‐induced inactivation phenomena attributable to neither of these effects are shown to occur in monomeric enzymes. Three different types of pressure dependence can be distinguished: (1) a linear dependence of catalytic rate constants on pressure, as predicted by the activated complex theory, observed for lysozyme and thermolysin; (2) a biphasic profile consisting of two linear contributions, found for trypsin; (3) maximum curves, as observed for both directions of the octopine dehydrogenase reaction. The third case may be ascribed to a pressure‐induced decrease in the partial specific volume of the protein, resulting in reduced flexibility of the active site. This mechanism may also apply to the pressure‐induced inactivation of assembly systems stabilized against dissociation in the cell.

Structure and Properties of an Engineered Transketolase from Maize

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.

Pressure dependence of collagen melting

Calf skin collagen type I and interstitial collagen of the annelids Alvinella pompejana and Riftia pachyptila were thermally unfolded at pressures of 1 and 200 bar. The high pressure was near the habitat pressure of the annelids which live in deep sea hydrothermal vents. The transition temperature increased with pressure by only 1.4 +/- 1 degrees C for calf skin collagen, and no pressure effect was detectable for the annelid collagens. The value for calf skin collagen agrees with prediction based on published values of the transition volume and transition enthalpy. The triple helices of the interstitial collagens of the annelids, which have melting temperatures of 46 degrees C (Alivinella pompejana) and 29 degrees C (Riftia pachyptila), are not further stabilized by pressure.

Studies on the Reaction Mechanism of GTP Cyclohydrolase I

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP (1, 2). The reaction involves the release of C-8 of GTP as formate. The catalytic domain of the human enzyme shows 37% identity with that of Escherichia coli. Both enzymes are toroid-shaped homodecamers with d5 symmetry and were recently shown to contain a catalytically essential zinc ion at each of the 10 equivalent active sites (Figure 1) (3, 4).