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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.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)

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Evolution of the genetic code in an early RNA world is dependent on the steadily improving specificity of the coevolving protein synthesis machinery for codons, anticodons, tRNAs and amino acids. In the beginning, there is RNA but the machinery does not distinguish yet between the codons, which therefore all encode the same information. Synonymous codons are equivalent under a symmetry group that exchanges (permutes) the codons without affecting the code. The initial group changes any codon into any other by permuting the order of the bases in the triplet as well as by replacing the four RNA bases with each other at every codon position. This group preserves the differences between codons, known as Hamming distances, with a 1-distance corresponding to a single point mutation. Stepwise breaking of the group into subgroups divides the 64 codons into progressively smaller subsets - blocks of equivalent codons under the smaller symmetry groups, with each block able to encode a different message. This formalism prescribes how the evolving machinery increasingly differentiates between codons. The model indicates that primitive ribosomes first identified a unique mRNA reading frame to break the group permuting the order of the bases and subsequently enforced increasingly stringent codon-anticodon basepairing rules to break the subgroups permuting the four bases at each codon position. The modern basepairing rules evolve in five steps and at each step the number of codon blocks doubles. The fourth step generates 16 codon blocks corresponding with the 16 family boxes of the standard code and the last step splits these boxes into 32 blocks of commonly two, but rarely one or three, synonymous codons. The evolving codes transmit at most one message per codon block and as the number of messages increases so does the specificity of the code and of protein synthesis. The selective advantage conferred by better functioning proteins drives the symmetry breaking process. Over time paralogous tRNA evolution expands the anticodon repertoire, which is divided into anticodon blocks matching the codon blocks under the stage-specific ribosomal basepairing rules. Contemporaneously an expanding family of primitive aminoacyl-tRNA synthetases (aaRSs) divides the tRNA diversities into various different and overlapping subsets: each aaRS accepts some tRNAs but rejects all others and several aaRSs may accept the same tRNA species. Selection favoring less ambiguous codes eliminates these overlaps and also imposes the ribosomal anticodon block division as ambiguity arises when different aaRSs accept tRNAs of the same anticodon block. Only when the tRNAs of one or several anticodon blocks are accepted by a unique aaRS does the code become specific. This coding pattern is observed in the standard code and the evolution of amino acid assignments by primitive aaRSs onto tRNAs is traced back via tRNA trees that picture a gradual division of tRNA diversities into blocks with increasingly specific amino acid assignments. Symmetry breaking combined with...