Abstract A cDNA encoding insulin-degrading enzyme (IDE) was cloned from tomato (Solanum lycopersicum) and expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase. GST-SlIDE was characterized as a neutral thiol-dependent metallopeptidase with insulinase activity: the recombinant enzyme cleaved the oxidized insulin B chain at eight peptide bonds, six of which are also targets of human IDE. Despite a certain preference for proline in the vicinity of the cleavage site, synthetic peptides were cleaved at apparently stochastic positions indicating that SlIDE, similar to IDEs from other organisms, does not recognize any particular amino acid motif in the primary structure of its substrates. Under steady-state conditions, an apparent K m of 62±7 μm and a catalytic efficiency (k cat/K m) of 62±15 mm -1 s-1 were determined for Abz-SKRDPPKMQTDLY(NO3)-NH2 as the substrate. GST-SlIDE was effectively inhibited by ATP at physiological concentrations, suggesting regulation of its activity in response to the energy status of the cell. While mammalian and plant IDEs share many of their biochemical properties, this similarity does not extend to their function in vivo, because insulin and the β-amyloid peptide, well-established substrates of mammalian IDEs, as well as insulin-related signaling appear to be absent from plant systems.

12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.

The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1ΔAVC,C6S), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys6 mutant lacked the NH2-terminal Ala–3-Val–2-Cys–1 (ΔAVC) extension present in some SP-A1 isoforms. SP-A1ΔAVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1ΔAVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1ΔAVC,C6S showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The Tm was 32.7 °C for SP-A1ΔAVC,C6S and 44.5 °C for SP-A1. Although SP-A1ΔAVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant membranes, and to undergo self-association in the presence of Ca2+. On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1ΔAVC,C6S to inhibit the production of tumor necrosis factor-α by macrophage-like U937 cells stimulated with either smooth or rough lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation. The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.

A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) fromArabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein inEscherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of α,β-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest withN-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of theLeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone. Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration withpara-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR.