Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni 2+

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs inEscherichia coli showed that LeAOS andLeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C18 fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS andLeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPLexpression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression ofLeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants. Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed 9-and 13-hydroperoxides of linoleic and linolenic acid to ␣-ketol, ␥-ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3 was Ϸ10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling. These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.Oxylipins comprise a group of biologically active compounds that are produced by oxidative metabolism of polyunsaturated fatty acids. Members of the eicosanoid family of lipid mediators have been studied extensively with respect to their biosynthesis from arachidonic acid and their function in diverse physiological processes in animal cells (1). In plants, which lack arachidonic acid, oxygenated derivatives of C 18 fatty acids participate in the regulation of many defense-related and developmental processes. The biosynthesis of most phytooxylipins is initiated by lipoxygenase (LOX), 1 which adds molecular oxygen to either the C-9 or C-13 position of linolenic or linoleic acid (2). The resulting hydroperoxides are further metabolized by several enzymes including three closely related members of the CYP74 family of cytochromes P-450: allene oxide synthase (AOS), hydroperoxide lyase (HPL), and divinyl ether synthase (DES). Indeed, much of the structural and functional diversity in oxylipin metabolism in plants can be accounted for by the activity of CYP74s that metabolize 9-and 13-hydroperoxides to a wide range of products (3). In contrast to typical P-450 monooxygenases, CYP74 P-450s do not require O 2 and a NADPHdependent P-450 reductase for activity. Rather, they use a hydroperoxide group both as the oxygen donor and as a source of reducing equivalents (4, 5). This unique catalytic feature is shared by thromboxane synthase and prostacyclin synthase, two P-450 enzymes involved in the synthesis of eicosanoids (6). A greater understanding of the biochemical and physiological function of this atypical class of P-450 enzymes promises to provide new insight into the evolution ...

Lipoxygenase-derived fatty acid hydroperoxides are metabolized by CYP74 cytochrome P-450s to various oxylipins that play important roles in plant growth and development. Here, we report the characterization of a Lycopersicon esculentum (tomato) cDNA whose predicted amino acid sequence defines a previously unidentified P-450 subfamily (CYP74D). The recombinant protein, expressed in Escherichia coli, displayed spectral properties of a P-450. The enzyme efficiently metabolized 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid but was poorly active against the corresponding 13-hydroperoxides. Incubation of recombinant CYP74D with 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid yielded divinyl ether fatty acids (colneleic acid and colnelenic acid, respectively), which have been implicated as plant anti-fungal toxins. This represents the first identification of a cDNA encoding a divinyl ether synthase and establishment of the enzyme as a CYP74 P-450. Genomic DNA blot analysis revealed the existence of a single divinyl ether synthase gene located on chromosome one of tomato. In tomato seedlings, root tissue was the major site of both divinyl ether synthase mRNA accumulation and enzyme activity. These results indicate that developmental expression of the divinyl ether synthase gene is an important determinant of the tissue specific synthesis of divinyl ether oxylipins.

No abstract

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization—GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

Herein we characterize the Arabidopsis thaliana AtLOX1 and tomato (Solanum lycopersicum) LOXA proteins as linoleate 9S-lipoxygenases (9-LOX), and use the enzymes to test a model that predicts a relationship between substrate binding orientation and product stereochemistry. The cDNAs were heterologously expressed in E. coli and the proteins partially purified by nickel affinity chromatography using a N-terminal (His)6-tag. Both enzymes oxygenated linoleic acid almost exclusively to the 9S-hydroperoxide with turnover numbers of 300–400/s. AtLOX1 showed a broad range of activity over the range pH 5–9 (optimal at pH 6); tomato LOXA also showed optimal activity around pH 5–7 dropping off more sharply at pH 9. Site-directed mutagenesis of a conserved active site Ala (Ala562 in AtLOX1, Ala 564 in tomato LOXA, and typically conserved as Ala in S-LOX and Gly in R-LOX), revealed that substitution with Gly led to the production of a mixture of 9S- and 13R-hydroperoxyoctadecadienoic acids from linoleic acid. To follow up on earlier reports of 9-LOX metabolism of anandamide (van Zadelhoff et al., 1998, Biochem. Biophys. Res. Commun. 248, 33), we also tested this substrate with the mutants, which produced predictable shifts in product profile, including a shift from the prominent 11S-hydroperoxy derivative of wild-type to include the 15R-hydroperoxide. These results conform to a model that predicts a head-first substrate binding orientation for 9S-LOX. We also found that linoleoyl-phosphatidylcholine is not a 9S-LOX substrate, which is consistent with this conclusion.

The SCO2299gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N‐terminal region shows high amino acid sequence similarity to RNase HI, whereas its C‐terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature‐sensitive growth defect of an Escherichia coli RNase H‐deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA·DNA hybrid in vitro. The N‐terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C‐terminal domain showed no CobC‐like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C‐terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N‐terminal and C‐terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.