Summary 12-Oxophytodienoate reductases (OPRs) belong to a family of¯avin-dependent oxidoreductases. With two new tomato isoforms reported here, three OPRs have now been characterized in both tomato and Arabidopsis. Only one of these isoforms (OPR3) participates directly in the octadecanoid pathway for jasmonic acid biosynthesis, as only OPR3 reduces the 9S,13S-stereoisomer of 12-oxophytodienoic acid, the biological precursor of jasmonic acid. The subcellular localization of OPRs was analyzed in tomato and Arabidopsis. The OPR3 protein and activity were consistently found in peroxisomes where they co-localize with the enzymes of b-oxidation which catalyze the ®nal steps in the formation of jasmonic acid. The octadecanoid pathway is thus con®ned to plastids and peroxisomes and, in contrast to previous assumptions, does not involve the cytosolic compartment. The expression of tomato (Lycopersicon esculentum, Le) OPR3 was analyzed in the context of defense-related genes using a microarray comprising 233 cDNA probes. LeOPR3 was found to be up-regulated after wounding with induction kinetics resembling those of other octadecanoid pathway enzymes. In contrast to the induction of genes for wound response proteins (e.g. proteinase inhibitors), the accumulation of octadecanoid pathway transcripts was found to be more rapid and transient in wounded leaves, but hardly detectable in unwounded, systemic leaves. Consistent with the expression data, OPDA and JA were found to accumulate locally but not systemically in the leaves of wounded tomato plants. The transcriptional activation of the octadecanoid pathway and the accumulation of JA to high levels are, thus not required for the activation of defense gene expression in systemic tissues.

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-A1 ⌬AVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys 6 mutant lacked the NH 2 -terminal Ala ؊3 -Val ؊2 -Cys ؊1 (⌬AVC) extension present in some SP-A1 isoforms. SP-A1⌬AVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1 ⌬AVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1 ⌬AVC,C6Sshowed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The T m was 32.7°C for SP-A1 ⌬AVC,C6S and 44.5°C for SP-A1. Although SP-A1 ⌬AVC,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 Ca 2؉ . On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1 ⌬AVC,C6S to inhibit the production of tumor necrosis factor-␣ by macrophagelike 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.

Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that alpha,beta-unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9R,13R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9R,13R-OPDA or a structurally related oxylipin as natural substrate of OPR1.

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.flavoprotein ͉ jasmonic acid biosynthesis ͉ plant defense ͉ oxylipin signature ͉ 12-oxophytodienoic acid F lavoproteins catalyze a wide variety of essential biochemical reactions, including electron transfer, dehydrogenation, and hydroxylation reactions. Old yellow enzyme (OYE), the first flavin-dependent enzyme identified (1), and homologues of OYE from bacteria, yeast, and plants are able to reduce the CAC double bond of ␣,␤-unsaturated carbonyl compounds, an activity that is rather uncommon for flavoenzymes (2, 3). This reaction has been shown to proceed by a ping-pong bi-bi mechanism, during which the FMN cofactor is reduced by NAD(P)H before the substrate is bound and reduced by hydride transfer to the substrate's C ␤ (4). Despite extensive efforts, the physiological substrate has been revealed only for one member of the OYE family, the plant enzyme 12-oxophytodienoate reductase (OPR) 3, which catalyzes one step in the biosynthesis of the plant hormone jasmonic acid (JA) (5, 6). JA and JA derivatives act as signaling compounds in the defense response against herbivores and pathogens and are involved in the regulation of various developmental processes, such as fruit ripening, pollen maturation, and senescence (7,8). JA is synthesized from ␣-linolenic acid, which is oxidized and cyclized, resulting in the cyclopentenone 9S,13S-12-oxophytodienoate (9S,13S-OPDA). OPR3 reduces 9S,13S-OPDA to the corresponding cyclopentanone, which is converted to JA by repeated ␤-oxidation ( Fig. 1) (9). Several OPR isozymes have been identified in plants, including 3 isoforms in Lycopersicon esculentum, 5 in Arabidopsis thaliana, and 13 in Oryza sativa (10, 11). O...

The alternative sigma factor B of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative A -dependent promoter, termed sigB p1 , giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a B -dependent promoter, sigB p3 , initiating a 1.6-kb transcript covering rsbV-rsbWsigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB p2 and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB p1 and sigB p3 . In vitro, the sigB p1 promoter was active in the early growth stages, while the sigB p2 and sigB p3 promoters produced transcripts throughout the growth cycle, with sigB p3 peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of B measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-B were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the B activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of B , however, signals a relevant difference in the regulation of B activation between B. subtilis and S. aureus in the cascade upstream of RsbU.Staphylococcus aureus is one of the leading causes for nosocomial-and community-acquired infections (11,46). Its capacity to cause a wide spectrum of diseases and to survive in unfavorable conditions is due to a network of global regulatory elements enabling it to rapidly sense changes and to respond appropriately. These elements comprise two-component regulatory systems, including the agr locus, the SarA protein family, and alternative factors (reviewed in reference 16 and references within).Computational analysis of the published staphylococcal genomes suggests that S. aureus harbors only two alternative sigma factors, B and H (45). B of S. aureus was demonstrated to influence the expression of a variety of genes (6,26,33,43,78,79), including virulence factors (23,27,34,38,43,50,51,52,62,78,79) and regulatory elements (5,6,21,26,34,47,60,66). Moreover, it affects methicillin and glycopeptide resistance (4,56,65,74), biofilm production (58), and internalization into endothelial ce...

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) from Arabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein in Escherichia 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 ␣,␤-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest with N-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of the LeOPR-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 with para-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.

We have identified imidazopyridine derivatives as a novel class of NO synthase inhibitors with high selectivity for the inducible isoform. 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo [4,5-b]pyridine (BYK191023) showed half-maximal inhibition of crudely purified human inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NO synthases at 86 nM, 17 M, and 162 M, respectively. Inhibition of inducible NO synthase was competitive with L-arginine, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. In radioligand and surface plasmon resonance experiments, BYK191023 exhibited an affinity for iNOS, nNOS, and eNOS of 450 nM, 30 M, and Ͼ500 M, respectively. Inhibition of cellular nitrate/nitrite synthesis in RAW, rat mesangium, and human embryonic kidney 293 cells after iNOS induction showed 40-to 100-fold higher IC 50 values than at the isolated enzyme, in agreement with the much higher L-arginine concentrations in cell culture media and inside intact cells. BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations. The inhibitory potency of BYK191023 was tested in isolated organ models of iNOS (lipopolysaccharide-treated and phenylephrine-precontracted rat aorta; IC 50 ϭ 7 M), eNOS (arecaidine propargyl esterinduced relaxation of phenylephrine-precontracted rat aorta; IC 50 Ͼ 100 M), and nNOS (field-stimulated relaxation of phenylephrine-precontracted rabbit corpus cavernosum; IC 50 Ͼ 100 M). These data confirm the high selectivity of BYK191023 for iNOS over eNOS and nNOS found at isolated enzymes. In summary, we have identified a new highly selective iNOS inhibitor structurally unrelated to known compounds and L-arginine. BYK191023 is a valuable tool for the investigation of iNOS-mediated effects in vitro and in vivo.NO synthases are enzymes responsible for the generation of nitric oxide from the amino acid L-arginine (for review, see Alderton et al., 2001). Two classes of enzymes exist, which differ in their activation profile and their capacity to generate NO. Once expressed the inducible NO synthase (iNOS) is active for prolonged periods and produces micromolar concentrations of NO over longer periods. The iNOS expression is stimulated in various cell types by proinflammatory signals and is involved in immune defense against invading pathogens (Stuehr et al., 1991;Schmidt and Walter, 1994;Moncada and Higgs, 1995). The constitutively expressed enThese studies were supported in part by grants SFB 553 C15 and SFB 432 B6 from the Deutsche Forschungsgemeinschaft (to S.S.).Article, publication date, and citation information can be found at

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase and was purified from bacterial extracts. LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Proline is unique among the proteinogenic amino acids in that its side chain is bonded to both the ␣-carbon and the amino group. The resulting cyclic structure imposes conformational restraints on proline-containing peptides relevant for structure and function of many physiologically important biomolecules. A key role for proline residues is the protection against nonspecific proteolytic degradation. Hence proline is frequently found and conserved in peptide hormones, neuropeptides, and growth factors (1-3). Many bioactive polypeptides share a Xaa-Pro motif at their N termini shielding them against nonspecific N-terminal degradation. The degradation of these peptides requires proteases with specificity for the Xaa-Pro motif including proline-selective dipeptidases (dipeptidyl peptidases II and IV, cleaving the post-Pro bond) and aminopeptidase P (Xaa-Pro aminopeptidase, cleaving the pre-Pro bond). Cleavage of the Xaa-Pro motif by either one of these peptidases may initiate the proteolytic degradation/inactivation of the peptide or may result in an altered bioactivity (2-4).Aminopeptidase P (APP, 1 EC 3.4.11.9) was first isolated from Escherichia coli (5) and has subsequently been characterized from many microbial and mammalian sources (reviewed in Ref. 4). Mammalian APPs are now known to comprise at least two distinct forms, a cytosolic form and a membrane-bound form attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (6 -14). APPs hydrolyze the peptide bond between any amino acid and a penultimate proline residue at the N termini of oligopeptide and protein substrates. A free amino group is required at the N terminus and the scissile bond must be in the trans configuration (15). The hydrolysis of dipeptides is very slow compared with the hydrolysis of longer chains, indicating the existence of a third subsite for substrate binding, which was confirmed for E. coli and mammalian APPs (10, 15). Likely physiological substrates of APP include bradykinin, substance P, and peptide-YY (15-18), and APP has been implicated in the regulation of cardiovascular and pulmonary functions in vivo (19 -21).In higher plants, only very few peptides with hormone-like functions are presently known (22, 23), but a more general role for peptides as signal molecu...