Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric-oxide synthase reductase domain reveal structural and functional differences.

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share approximately 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.

Use of mechanism-based structure-activity relationships analysis in carcinogenic potential ranking for drinking water disinfection by-products.

Disinfection by-products (DBPs) are formed when disinfectants such as chlorine, chloramine, and ozone react with organic and inorganic matter in water. The observations that some DBPs such as trihalomethanes (THMs), di-/trichloroacetic acids, and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) are carcinogenic in animal studies have raised public concern over the possible adverse health effects of DBPs. To date, several hundred DBPs have been identified. To prioritize research efforts, an in-depth, mechanism-based structure-activity relationship analysis, supplemented by extensive literature search for genotoxicity and other data, was conducted for ranking the carcinogenic potential of DBPs that met the following criteria: a) detected in actual drinking water samples, b) have insufficient cancer bioassay data for risk assessment, and c) have structural features/alerts or short-term predictive assays indicative of carcinogenic potential. A semiquantitative concern rating scale of low, marginal, low-moderate, moderate, high-moderate, and high was used along with delineation of scientific rationale. Of the 209 DBPs analyzed, 20 were of priority concern with a moderate or high-moderate rating. Of these, four were structural analogs of MX and five were haloalkanes that presumably will be controlled by existing and future THM regulations. The other eleven DBPs, which included halonitriles (6), haloketones (2), haloaldehyde (1), halonitroalkane (1), and dialdehyde (1), are suitable priority candidates for future carcinogenicity testing and/or mechanistic studies.

Physiochemical Characterization and Field Assessment of Lettuce Chlorosis Virus.

Lettuce chlorosis virus (LCV) was purified and partially characterized, and polyclonal antisera were produced and used to assess disease in the field. The antisera reliably detected LCV by indirect enzyme-linked immunosorbent assay (ELISA) in Nicotiana benthamiana. In Western blots, the LCV antisera distinguished between LCV and lettuce infectious yellows virus (LIYV)-infected plants. LCV particle lengths in partially purified preparations, as observed by transmission electron microscopy, were variable, with the majority between 750 and 950 nm long. A single, high molecular weight dsRNA and several lower molecular weight dsRNAs were isolated from LCV-infected N. benthamiana. A single RNA isolated from purified virion preparations was estimated to be 8,625 nucleotides long and was suspected to be the genomic RNA of LCV. LCV was present in experimental field plots in Holtville, California, during the lettuce growing seasons of 1995 to 1997. The percentage of symptomatic plants and yield of lettuce heads treated with insecticide, as well as dsRNA and ELISA reactions for the plots, are reported. A dsRNA consistent in size with LCV was isolated from four weed species in the Imperial Valley of California.