Benastatin A

The entire gene locus encoding the biosynthesis of the potent glutathione-S-transferase inhibitors and apoptosis inducers benastatin A and B has been cloned and sequenced. The cluster identity was unequivocally proven by deletion of flanking regions and heterologous expression in S. albus and S. lividans. Inactivation and complementation experiments revealed that a KSIII component (BenQ) similar to FabH is crucial for providing and selecting the rare hexanoate PKS starter unit. In the absence of BenQ, several novel penta- and hexacyclic benastatin derivatives with antiproliferative activities are formed. In total, five new compounds were isolated and fully characterized, and the chemical analysis was confirmed by derivatization. The most intriguing observation is that the ben PKS can utilize typical straight and branched fatty acid synthase primers. If shorter straight-chain starters are utilized, the length of the polyketide backbone is increased, resulting in the formation of an extended, hexacyclic ring system reminiscent of proposed intermediates in the griseorhodin and fredericamycin pathways. Analysis and manipulation of the hybrid fatty acid polyketide pathway provides strong support for the hypothesis that the number of chain elongations is dependent on the total size of the polyketide chain that is accommodated in the PKS enzyme cavity. Our results also further substantiate the potential of metabolic engineering toward polyphenols with altered substituents and ring systems.

A glutathione S-transferase (GST) related to the phi (F) class of enzymes only found in plants has been cloned from the Oryza sativa. The GST cDNA was cloned by PCR using oligonucleotide primers based on the OsGSTF5 (GenBank Accession No. AF309382) sequences. The cDNA was composed of a 669-bp open reading frame encoding for 223 amino acids. The deduced peptide of this gene shared on overall identity of 75% with other known phi class GST sequences. On the other hands, the OsGSTF5 sequence showed only 34% identity with the sequence of the OsGSTF3 cloned by our previous study (Cho et al., 2005). This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF5 formed a homo-dimer composed of 28 kDa subunit and its pI value was approximately 7.8. The expressed OsGSTF5 displayed glutathione conjugation activity toward 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane and glutathione peroxidase activity toward cumene hydroperoxide. The OsGSTF5 also had high activities towards the herbicides alachlor, atrazine and metolachlor. The OsGSTF5 was highly sensitive to inhibition by ShexylGSH, benastatin A and hematin. We propose from these results that the expressed OsGSTF5 is a phi class GST and appears to play a role in the conjugation of herbicide and GPOX activity.

The benastatins, pradimicins, fredericamycins, and members of the griseorhodin/rubromycin family represent a structurally and functionally diverse group of long-chain polyphenols from actinomycetes. Comparison of their biosynthetic gene clusters (ben, prm, fdm, grh, rub) revealed that all loci harbor genes coding for a similar, yet uncharacterized, type of ketoreductases. In a phylogenetic survey of representative KRs involved in type II PKS systems, we found that it is generally possible to deduce the KR regiospecificity (C-9, C-15, C17) from the amino acid sequence and thus to predict the nature of the aromatic polyketide (e.g., angucycline, anthracycline, benzoisochromanequinones). We hypothezised that the new clade of KRs is characteristic for biosynthesis of polyphenols with an extended angular architecture we termed "pentangular". To test this hypothesis, we demonstrated the biogenetic relationship between benastatin and the structurally unrelated spiro ketal griseorhodin by generating a mutant producing collinone, a pentangular pathway intermediate. The benastatin pathway served as a model to characterize the KR. Gene inactivation of benL resulted in the formation of a series of 19-hydroxy benastatin and bequinostatin derivatives (e.g., benastatin K and benastatin L). These results clearly showed that BenL functions as a C-19 KR in pentangular pathways.