6: [1][2][3][4][5][6][7][8][9][10][11] 1987]). Using antibodies directed against cytochrome P-450su1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450su1. Similar information about the cytochrome P-45OSU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2. The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-45OcA, from Pseudomonas putida. Both cytochromes P-450su1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-45OcAm, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group.Streptomyces griseolus ATCC 11796 is capable of metabolizing a number of sulfonylurea herbicides to compounds that often exhibit reduced phytotoxicity (23,24,30). It does so via two sulfonylurea-inducible cytochrome P-450 monooxygenases designated cytochrome P-450su1 and cytochrome P-450SU2 (24). Partial characterization and reconstitution studies (23,24) suggest that the two inducible P-450 monooxygenase systems of S. griseolus resemble the threecomponent cytochrome P-450CAM camphor oxidation system of Pseudomonas putida ATCC 17453 (12). In the P-450CAM system, the genes for the three components, putidaredoxin reductase, putidaredoxin (iron-sulfur protein), and cytochrome P-450c,m, have been designated camA, camB, and camC, respectively (15,28). In line with this genetic nomenclature and anticipating the genetic characterization of all the components of the S. griseolus systems, we propose to name the gene for cytochrome P-450su1 suaC and the gene for cytochrome P-450SU2 subC.We are interested in analyzing the genes for these sulfonylurea-metabolizing systems and how they are regulated to understand how a soil organism responds to and metabolizes chemicals foreign to its environment. Additionally, we would like to introduce these genes into plants to enable them to metabolize sulfonylureas to less phytotoxic compounds. In this study, we used the characteristics of the cytochrome P-450su1 and P-450SU2 apoproteins (amino acid composition, NH2-terminal sequence, and antigenicity) and their relatedness to one another to identify and clone the * Corresponding author. t Du Pont Experimental Station Contribution 5241.

The Sfreptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450sul (CYP105Al) was expressed in transgenic tobacco (Nicofiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450sul antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450sul-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl~-1,2-benzoisothiazole-7-sulfonamide-1,l-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450sul are susceptible to injury from R7402 treatment that is harmless to plants without P45OSul. Thus, P450sul expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450sul from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450sul from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent.P450 monooxygenases play a central role in plant response to a variety of environmental challenges, including herbicide and other pesticide treatments (Cole, 1983;Durst, 1991). The best examples of this role are those P450s that can chemically alter an herbicide to a form with reduced phytotoxicity, resulting in much higher herbicide tolerance in the plant where this metabolism occurs (Frear et al., 1969(Frear et al., ,1991Sweetser et al., 1982; Jacobson and Shimabukuru, 1984;Brown, 1990). Significant variability in P450 enzyme activity exists among plant species, both in the presence of distinct enzymic forms and in their range of substrate specificity. This variability is a major determinant in herbicide selectivity, the differential herbicide sensitivity of weed and crop species. Altering herbicide selectivity, particularly confemng resistance in a crop plant, might be accomplished by expression of Present address:

The microsomal fraction from the mesocarp of avocado (Persea amerkana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest pattems, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarites with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.Cytochrome P450 dependent monooxygenases are widespread in nature. They are involved in a variety of catabolic and biosynthetic pathways and in the metabolism of drugs and xenobiotics. In plants, Cyt P-450 has been identified, for example, in the trans-cinnamic acid and kaurene hydroxylases (10,11). Others are suspected participants in a number of reactions associated with xenobiotic metabolism (6, 8) (for reviews see Refs. 4 and 20). Within these monooxygenase systems, Cyt P-450 serves as a terminal oxidase, responsible for substrate recognition, binding, and oxygen redox chemistry (5, 29).The mechanistic and structural details of Cyt P450 dependent monooxygenases have been developed largely from studies of the bacterial (Pseudomonas putida) camphor hydroxylase system (23,29). Because of their importance in xenobiotic and drug metabolism, these enzymes have also been thoroughly studied in mammalian liver (3,5,31). In contrast, Cyt P-450 of higher plant origin has not been the subject of extensive biochemical characterization, primarily because of the low content in many plant tissues, difficulties with identification in Chl containing tissues, and supposed lability in homogenized plant extracts. ' (6,10,12,13,19,20). Cyt P-450 has been purified from tulip (Tulipa gesneriana) bulbs (12), and from tubers of Jerusalem artichoke (Helianthus tuberosus) (10). Additionally, an NADPH:Cyt P450 reductase has been purified from Jerusalem artichoke (2), and the related Cyt b5 and NADH:Cyt b5 reductase have been purified from Pisum sativum (16). Unfortunately, the tulip bulb Cyt P450 has not been associated with any enzymic activity, and polyclonal antibodies raised against it were not cross reactive with proteins from other species (1 1). The Jerusalem artichoke preparation can be reconstituted into its trans-cinnamate hydroxylase activity, but it has a low heme specific content and is not homogeneous (10). No primary sequence information has been reported for either o...

We have studied the synthesis and accumulation of a chloroplast‐encoded 48 kd chla‐reaction center protein and the 34.5 kd ‘atrazine binding’ protein in a nuclear maize mutant which fails to assemble photosystem II reaction centers. The failure of these polypeptides to accumulation in mutant thylakoids is not due to direct nuclear control over their synthesis but is rather due to their specific, accelerated turnover from the thylakoid membrane. The accelerated turnover of these polypeptides in mutant thylakoids is largely independent of illumination conditions, as accelerated turnover occurs in the dark as well as in the light. In contrast to wild type, the 48 kd and 34.5 kd polypeptides are preferentially associated with stroma, rather than grana, lamellae in mutant membranes, suggesting that turnover occurs before these polypeptides become enriched in the grana. The nucleus thus plays a role in the stabilization of these chloroplast‐encoded photosystem II reaction center polypeptides.

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