Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents

Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450_(BM3) for Selective Hydroxylation of Large Substrates

Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_(BM3) variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates

Arabidopsis SUC1 loads the phloem in suc2 mutants when expressed from the SUC2 promoter

Active loading of sucrose into phloem companion cells (CCs) is an essential process in apoplastic loaders, such as Arabidopsis or tobacco (Nicotiana sp.), and is even used by symplastic loaders such as melon (Cucumis melo) under certain stress conditions. Reduction of the amount or complete removal of the transporters catalysing this transport step results in severe developmental defects. Here we present analyses of two Arabidopsis lines, suc2-4 and suc2-5, that carry a null allele of the SUC2 gene which encodes the Arabidopsis phloem loader. These lines were complemented with constructs expressing either the Arabidopsis SUC1 or the Ustilago maydis srt1 cDNA from the SUC2 promoter. Both SUC1 and Srt1 are energy-dependent sucrose/H+ symporters and differ in specific kinetic properties from the SUC2 protein. Transgene expression was confirmed by RT-PCRs, the subcellular localization of Srt1 in planta with an Srt1-RFP fusion, and the correct CC-specific localization of the recombinant proteins by immunolocalization with anti-Srt1 and anti-SUC1 antisera. The transport capacity of Srt1 was studied in Srt1-GFP expressing Arabidopsis protoplasts. Although both proteins were found exclusively in CCs, only SUC1 complemented the developmental defects of suc2-4 and suc2-5 mutants. As SUC1 and Srt1 are well characterized, this result provides an insight into the properties that are essential for sucrose transporters to load the phloem successfully

Recombinatorial and Predictive Methods to Increase Cellulase Thermostability and Structural Analysis of a Thermostable P450

To address the world’s need for improved biomass breakdown for the production of renewable fuel, we sought to improve cellulase thermostability and thereby enzyme lifetime, operating temperature, and specific activity. We created an eight block SCHEMA recombination library based on five fungal cellobiohydrolase class I (CBHI) enzymes. By characterizing this library, we identified several stabilizing sequence blocks and combined these to produce a set of well-expressed, thermostable CBHI chimeras. To further increase the stability of these chimeras, we used a combination of the chimera thermostability screening data, a consensus analysis of 40 naturally occurring CBHI sequences, and FoldX ΔΔG predictions to identify individual mutations for testing. Our final enzyme has a T50 9.3 °C greater than that of the most stable parental CBHI, resulting in a 10 °C increase in optimal temperature and a 50% increase in total sugar production at the optimal temperature. To produce an ideal parent for directed evolution for improved activity on varied compounds, we increased the thermostability of a P450BM3 enzyme with broad substrate specificity to produce enzyme 9-10ATS. Directed evolution libraries based on 9-10ATS produced variants with improved activity on a number of structurally diverse compounds. We determined the structure of 9-10ATS using x-ray crystallography and compared it to other P450BM3 structures. Examination of the stucture shows clear structural basis for the thermostabilizing mutations and broad substrate specificity.</p

Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods

Building on our previous efforts to generate thermostable chimeric fungal cellobiohydrolase I (CBH I, also known as Cel7A) cellulases by structure-guided recombination, we used FoldX and a ‘consensus’ sequence approach to identify individual mutations present in the five homologous parent CBH I enzymes which further stabilize the chimeras. Using the FoldX force field, we calculated the effect on ΔG_Folding of each candidate mutation in a number of CBH I structures and chose those predicted to be stabilizing in multiple structures. With an alignment of 41 CBH I sequences, we also used amino acid frequencies at each candidate position to calculate predicted effects on ΔG_Folding. A combination of mutations chosen using these methods increased the T_50 of the most thermostable chimera by an additional 4.7°C, to yield a CBH I with T_50 of 72.1°C, which is 9.2°C higher than that of the most stable native CBH I, from Talaromyces emersonii. This increased stability resulted in a 10°C increase in the optimal temperature for activity, to 65°C, and a 50% increase in total sugar production from crystalline cellulose at the optimal temperature, compared with native T.emersonii CBH I

Cell-selective activity of rhodium metalloinsertors correlates with subcellular localization

Deficiencies in the mismatch repair (MMR) pathway have been assocd. with various cancers, and many commonly used chemotherapeutics have decreased effectiveness against MMR-deficient cancers. There is consequently a need for chemotherapies that selectively target MMR-deficient cancer cells. Research in our lab. has uncovered a class of compds., rhodium metalloinsertors, that selectively binds to DNA mismatches with high affinity and specificity. These rhodium metalloinsertors bear an expansive arom. chrysi ligand (chrysi= chrysene-5,6-quinonediimine) which, upon mismatch binding, inserts into the DNA duplex from the minor groove and ejects the mismatched bases. These metalloinsertors exhibit selective cytotoxicity, preferentially inducing necrosis in MMR-deficient cells over MMR-proficient cells. They have been found to localize in the nuclei of MMR-deficient cells at concns. sufficient for mismatch detection, and their ability to selectively target MMRdeficient cells has been shown to be contingent upon low mitochondrial rhodium accumulation

Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination

We describe an efficient SCHEMA recombination-based approach for screening homologous enzymes to identify stabilizing amino acid sequence blocks. This approach has been used to generate active, thermostable cellobiohydrolase class I (CBH I) enzymes from the 390 625 possible chimeras that can be made by swapping eight blocks from five fungal homologs. Constructing and characterizing the parent enzymes and just 32 ‘monomeras’ containing a single block from a homologous enzyme allowed stability contributions to be assigned to 36 of the 40 blocks from which the CBH I chimeras can be assembled. Sixteen of 16 predicted thermostable chimeras, with an average of 37 mutations relative to the closest parent, are more thermostable than the most stable parent CBH I, from the thermophilic fungus Talaromyces emersonii. Whereas none of the parent CBH Is were active >65°C, stable CBH I chimeras hydrolyzed solid cellulose at 70°C. In addition to providing a collection of diverse, thermostable CBH Is that can complement previously described stable CBH II chimeras (Heinzelman et al., Proc. Natl Acad. Sci. USA 2009;106:5610–5615) in formulating application-specific cellulase mixtures, the results show the utility of SCHEMA recombination for screening large swaths of natural enzyme sequence space for desirable amino acid blocks