| A three-dimensional model of the Streptomyces coelicolor actinorhodin β-ketoacyl synthase (Act KS) was constructed based on the x-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme KAS II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. 3H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of amino acid replacements affected the formation of the active site nucleophile.;Detailed analysis of mitomycin C binding protein MRD has revealed that it reductively transforms MC into 1,2-cis-1-hydroxy-2,7-diaminomitosene, a product which is also generated in the reductive MC activation cascade. However, the reductive reaction rate is slow and both MC and the reduced product are protein-bound for a prolonged period. DNA shuffling technology, amino acid sequence analysis and site-directed mutational analysis have revealed that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase/peroxidases and is important for MRD function. Together, these findings support an evolutionary model suggesting that MRD has descended from a previously unidentified quinone reductase. Subsequent mutations at the active site resulted in attenuated catalytic activity while preserving the substratebinding capability, thus switching the protein from a potential drug activating enzyme into the drug-binding component of the MC export system.;The potential interaction between MRD and the drug-transporter MCT has been investigated in a heterologous expression system of E. coli and an in vitro protein crosslinking assay. The results suggested that functional cooperation between MRD and MCT may involve the dimeric form of each protein. The results of systematic disruption of MC resistance genetic loci (including mrd, mct and mcrA) in S. lavendulae have suggested that MRD is one of the predominant self-protection components in S. lavendulae. |
