Modification of small molecules by using cytochrome P450 expressed in Escherichia coli.

We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening.

Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

Cloning, expression in yeast, and functional characterization of CYP76A4, a novel cytochrome P450 of petunia that catalyzes (omega-1)-hydroxylation of lauric acid.

A cDNA clone of a novel cytochrome P450, CYP76A4, was isolated from Petunia hybrida. The cDNA clone contained an open reading frame (ORF) encoding a predicted 510 amino acid polypeptide. The CYP76A4 cDNA was expressed in yeast Saccharomyces cerevisiae AH22. Recombinant yeast microsomes containing the CYP76A4 hemoprotein were found to catalyze (omega-1)-hydroxylation of lauric acid.

Two novel mitogen-activated protein signaling components, OsMEK1 and OsMAP1, are involved in a moderate low-temperature signaling pathway in rice.

Rice (Oryza sativa) anther development is easily damaged by moderately low temperatures above 12 degrees C. Subtractive screening of cDNA that accumulated in 12 degrees C-treated anthers identified a cDNA clone, OsMEK1, encoding a protein with features characteristic of a mitogen-activated protein (MAP) kinase kinase. The putative OsMEK1 protein shows 92% identity to the maize (Zea mays) MEK homolog, ZmMEK1. OsMEK1 transcript levels were induced in rice anthers by 12 degrees C treatment for 48 h. Similar OsMEK1 induction was observed in shoots and roots of seedlings that were treated at 12 degrees C for up to 24 h. It is interesting that no induction of OsMEK1 transcripts was observed in 4 degrees C-treated seedlings. In contrast, rice lip19, encoding a bZIP protein possibly involved in low temperature signal transduction, was not induced by 12 degrees C treatment but was induced by 4 degrees C treatment. Among the three MAP kinase homologs cloned, only OsMAP1 displayed similar 12 degrees C-specific induction pattern as OsMEK1. A yeast two-hybrid system revealed that OsMEK1 interacts with OsMAP1, but not with OsMAP2 and OsMAP3, suggesting that OsMEK1 and OsMAP1 probably function in the same signaling pathway. An in-gel assay of protein kinase activity revealed that a protein kinase (approximately 43 kD), which preferentially uses myelin basic protein as a substrate, was activated by 12 degrees C treatment but not by 4 degrees C treatment. Taken together, these results lead us to conclude that at least two signaling pathways for low temperature stress exist in rice, and that a MAP kinase pathway with OsMEK1 and OsMAP1 components is possibly involved in the signaling for the higher range low-temperature stress.