Charles A. Omer

Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice.

For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.

Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P‐450soy (soyC) and ferredoxinsoy (soyB) from Streptomyces griseus

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P‐450, designated cytochrome P‐450soy A DNA segment carrying the structural gene encoding P‐450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P‐450soy was compared with that for P‐450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe‐4S] ferredoxin (soyB), named ferredoxinsoy, was identified.

Selection of Potent Inhibitors of Farnesyl-protein Transferase from a Synthetic Tetrapeptide Combinatorial Library

Inhibitors of farnesyl-protein transferase (FPTase) show promise as anticancer agents. Based on the sequence of the protein substrates of FPTase (the CAAX sequence), potent and selective peptidomimetic inhibitors have been developed; these compounds share with the peptide substrate a free thiol and a C-terminal carboxylate. We have used a synthetic tetrapeptide combinatorial library to screen for new leads devoid of these features: the peptides were C-terminally amidated, and no free thiol was included in the combinatorial building blocks. To compensate for this negative bias, an expanded set of 68 amino acids was used, including both L and D as well as many non-coded residues. Sixteen individual tetrapeptides derived from the consensus were synthesized and tested; all were active, showing IC50 values ranging from low micromolar to low nanomolar. The most active peptide, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-γ-carboxyglutamic acid (Ki = 2 nM), is also very selective showing little inhibitory activity against the related enzyme geranylgeranyl-protein transferase type I (IC50 > 50 μM). In contrast to CAAX-based peptidomimetics, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-γ-carboxyglutamic acid appeared to mimic the isoprenoid substrate farnesyl diphosphate as determined by kinetic and physical measurements. D-Tryptophan-Dmethionine-D-4-chlorophenylalanine-L-γ-carboxyglutamic acid was a competitive inhibitor of FPTase with respect to farnesyl diphosphate substrate and uncompetitive with respect to CAAX substrate. Furthermore, we demonstrated that FPTase undergoes ligand dependent conformational changes in its circular dichroism spectrum and that D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-γ-carboxyglutamic acid induced a conformational change identical to that observed with farnesyl diphosphate ligand.

[I] Bacterial expression and purification of human protein prenyltransferases using epitope-tagged, translationally coupled systems

Publisher Summary This chapter discusses the bacterial expression and purification of human protein prenyltransferases using epitope-tagged, translationally coupled systems. Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I), enzymes, which catalyze the transfer of a C 15 (FTase) or C 20 (GGTase I) isoprenoid to the carboxyl-terminal cysteine residue of proteins terminating with a CaaX sequence, are αβ heterodimers that share a common subunit—α. The single-plasmid systems developed for expression in Escherichia coli and subsequent rapid purification of human FTase and GGTase I are described in the chapter. The expression systems to produce site-directed mutants of FTase for structure–function analysis of the protein are used. Because the purification scheme does not require analyzing fractions for enzymatic activity, even catalytically inactive mutants can be purified for analysis. Heterodimeric proteins, such as FTase and GGTase are also expressed at high levels in baculovirus-infected insect cells and for eukaryotic proteins in which posttranslational modification is required for function insect cell expression can be advantageous.

Actinomycete cytochromes P‐450 involved in oxidative metabolism: Biochemistry and molecular biology∗

Abstract The diverse metabolic capability of actinomycetes, together with their widespread presence in terrestrial and aquatic environments, has rendered them instrumental in the breakdown of both man‐made and natural chemicals and in the recycling of carbon in nature. Actinomycetes are also well known for their ability to synthesize a wide variety of novel chemicals of pharmaceutical and industrial value. Cytochromes P‐450, a class of oxidative enzymes found in all organisms, play a central role in both biosynthetic and biodegradative reactions performed by actinomycetes. Herein, we describe recent studies of actinomycetes cytochromes P‐450 made possible by advances realized in biochemistry and molecular biology.

Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabolizing cytochromes P-450 in Streptomyces griseolus

We have identified the promoters for two inducible genes, in Streptomyces griseolus, that encode herbicide-metabolizing cytochromes P-450. They are in the class of promoters that have -35 and -10 sequences similar to those used in Escherichia coli by RNA polymerase E sigma 70. Transcription from either promoter was shown to be induced by sulfonylurea (chlorimuron ethyl) or phenobarbital. Mapping of mRNA showed that each cytochrome P450-encoding gene was transcribed on a separate multicistronic mRNA that encodes cytochrome P-450 (suaC or subC), ferredoxin (suaB or subB) and at least one other open reading frame. An inducible, site-specific DNA-binding activity was identified that bound to two similar 8-bp inverted repeat sequences within or near the sua promoter (suaP). A noninducible DNA-binding activity, distinct from that which bound to suaP, was found that bound to an 11-bp inverted repeat at the sub transcription start point.

Isolation and characterization of Streptomyces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism

SummaryMetabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450SU1 and P-450SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450SU2 in the absence of P-450SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.

Activation and detection of (Pro)mutagenic chemicals using recombinant strains ofStreptomyces griseus

Two recombinant strains ofStreptomyces griseus have been developed to report on the activation of promutagenic° chemicals. This activation is monitored by reversion of the bacterial test strains to a kana-mycin-resistant phenotype. Strain H69 detects point mutations and was reverted at an increased frequency by acetonitrile, 2-aminoanthracene, 1,2-benzanthracene, benzidine, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene, and glycine. The second strain, FS2, detects frame shift mutations and was reverted at an increased frequency by 1,2-benzanthracene, benzidine, and glycine. Compounds such as butylated hydroxytoluene, catechol, chlorobenzene, hydroquinone, potassium chloride, phenol,cis-stilbene,trans-stilbene, and toluene did not elicit positive responses in either strain. In addition, these strains are capable of detecting direct-acting mutagens such asN-methyl-N’-nitrosoguanidine and ICR-191, providing further evidence of their promise for detecting a wider range of mutagens. To our knowledge, this is the first report of bacterial strains capable of activating promutagenic compounds and detecting their mutagenic metabolites without the benefit of an exogenous activation system such as the rodent liver homogenate (S9).