Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.

A combined protein and pharmacophore model for cytochrome P450 2D6 (CYP2D6) has been extended with a second pharmacophore in order to explain CYP2D6 catalyzed N-dealkylation reactions. A group of 14 experimentally verified N-dealkylation reactions form the basis of this second pharmacophore. The combined model can now accommodate both the usual hydroxylation and O-demethylation reactions catalyzed by CYP2D6, as well as the less common N-dealkylation reactions. The combined model now contains 72 metabolic pathways catalyzed by CYP2D6 in 51 substrates. The model was then used to predict the involvement of CYP2D6 in the metabolism of a "test set" of seven compounds. Molecular orbital calculations were used to suggest energetically favorable sites of metabolism, which were then examined using modeling techniques. The combined model correctly predicted 6 of the 8 observed metabolites. For the well-established CYP2D6 metabolic routes, the predictive value of the current combined protein and pharmacophore model is good. Except for the highly unusual metabolism of procainamide and ritonavir, the known metabolites not included in the development of the model were all predicted by the current model. Two possible metabolites have been predicted by the current model, which have not been detected experimentally. In these cases, the model may be able to guide experiments. P450 models, like the one presented here, have wide applications in the drug design process which will contribute to the prediction and elimination of polymorphic metabolism and drug-drug interactions.

Cytochromes P450 (P450s) constitute a superfamily of phase I enzymes capable of oxidizing and reducing various substrates. P450 2D6 is a polymorphic enzyme, which is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. This deficiency leads to impaired metabolism of a variety of drugs. All drugs metabolized by P450 2D6 contain a basic nitrogen atom, and a flat hydrophobic region coplanar to the oxidation site which is either 5 or 7 A away from the basic nitrogen atom. The aim of this study was to build a three-dimensional structure for the protein and more specifically for the active site of P450 2D6 in order to determine the amino acid residues possibly responsible for binding and/ or catalytic activity. Furthermore, the structural features of the active site can be implemented into the existing small molecule substrate model, thus enhancing its predictive value with respect to possible metabolism by P450 2D6. As no crystal structures are yet available for membrane-bound P450s (such as P450 2D6), the crystal structures of bacterial (soluble) P450 101 (P450cam), P450 102 (P450BM3), and P450 108 (P450terp) have been used to build a three-dimensional model for P450 2D6 with molecular modeling techniques. Several important P450 2D6 substrates were consecutively docked into the active site of the protein model. The energy optimized positions of the substrates in the protein agreed well with the original relative positions of the substrates within the substrate model. This confirms the usefulness of small molecule models in the absence of structural protein data. Furthermore, the derived protein model indicates new leads for experimental validation and extension of the substrate model.

Cytochromes P450 (P450s) constitute a large superfamily of heme-containing enzymes, capable of oxidizing and reducing a variety of substrates. Cytochrome P450 2D6 is a polymorphic member of the P450 superfamily and is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. Recently, the importance of aspartic acid 301 (Asp301) for the catalytic activity of P450 2D6, as indicated by a preliminary homology model, was confirmed by site-directed mutagenesis experiments. In this study, the heme moiety and the I-helix containing Asp301 were incorporated into the previously derived substrate model for P450 2D6, in the spatial orientations as derived from a recently improved protein model for P450 2D6, thereby incorporating steric restrictions and orientational preferences into the substrate model. The direction of well-defined hydrogen bonds formed between Asp301 and basic nitrogen atoms of P450 2D6 substrates was incorporated into the substrate model as well. Also, the position(s) of the basic nitrogen atom(s) of the substrates was/were allowed more flexibility. This was established through the attachment of an aspartic acid residue (representing Asp301) to the (protonated) basic nitrogen atom(s) of the substrates and superimposing the C alpha- and C beta-atoms of this aspartic acid residue in the fitting procedure instead of the basic nitrogen atoms. A variety of 8 substrates of P450 2D6 (comprising 17 known P450 2D6 dependent metabolic pathways) has been incorporated successfully into this refined and more restrictive substrate model.

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3-10-fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts from recalcitrant plant species.