Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an Ester Bond-Forming Non-ribosomal Peptide Synthetase Module

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on themultifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique nonribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues

Crystal Structures of Acyl Carrier Protein in Complex with Two Catalytic Partners Show a Dynamic Role in Cellular Metabolism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107352/1/1079_ftp.pd

Pressure-induced hole doping of the Hg-based cuprate superconductors

We investigate the electronic structure and the hole content in the copper-oxygen planes of Hg based high Tc cuprates for one to four CuO2 layers and hydrostatic pressures up to 15 GPa. We find that with the pressure-induced additional number of holes of the order of 0.05e the density of states at the Fermi level changes approximately by a factor of 2. At the same time the saddle point is moved to the Fermi level accompanied by an enhanced k_z dispersion. This finding explains the pressure behavior of Tc and leads to the conclusion that the applicability of the van Hove scenario is restricted. By comparison with experiment, we estimate the coupling constant to be of the order of 1, ruling out the weak coupling limit.Comment: 4 pages, 4 figure

Characterization of the Β-Methylaspartate-Α-decarboxylase (CrpG) from the Cryptophycin Biosynthetic Pathway

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56158/1/1373_ftp.pd

Substrate Scope Analysis of Biocatalytic Halogenation on Complex Substrates

Malbrancheamide is a fungal natural product with significant vasorelaxation effects and potential as a cardiovascular therapeutic. The dichlorination of the indole ring is key for its biological activity, and this transformation is performed by the flavin dependent halogenase MalA. This enzyme utilizes a proposed chloramine lysine intermediate to iteratively and selectively chlorinate its natural substrate premalbrancheamide. Halogenases can provide orthogonal selectivity to many chemical methods, making them useful for pharmaceutical applications, while providing selective methods for late-stage functionalization. This investigation focuses on the substrate scope of the halogenase on complex pharmaceutically relevant substrates in collaboration with the Novartis Institutes for Biomedical Research. The bromination and chlorination reaction conditions were optimized, and the products were structurally characterized by NMR spectroscopy to gain further understanding of the versatility of the wild type enzyme and its mutants

Studies on the Biosynthesis of the Stephacidin and Notoamide Natural Products: A Stereochemical and Genetic Conundrum

The stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a bicyclo[2.2.2]diazaoctane core. Biosynthetically, this bicyclic core is believed to be the product of an intermolecular Diels–Alder (IMDA) cycloaddition of an achiral azadiene. Since all of the natural products in this family have been isolated in enantiomerically pure form to date, it is believed that an elusive Diels–Alderase enzyme mediates the IMDA reaction. Adding further intrigue to this biosynthetic puzzle is the fact that several related Aspergillus fungi produce a number of metabolites with the opposite absolute configuration, implying that these fungi have evolved enantiomerically distinct Diels–Alderases. We have undertaken a program to identify every step in the biogenesis of the stephacidins and notoamides, and by combining the techniques of chemical synthesis and biochemical analysis we have been able to identify the two prenyltransferases involved in the early stages of the stephacidin and notoamide biosyntheses. This has allowed us to propose a modified biosynthesis for stephacidin A, and has brought us closer to our goal of finding evidence for, or against, the presence of a Diels–Alderase in this biosynthetic pathway.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83757/1/442_ftp.pd

Substrate Scope Analysis of Biocatalytic Halogenation on Complex Substrates

Malbrancheamide is a fungal natural product with significant vasorelaxation effects and potential as a cardiovascular therapeutic. The dichlorination of the indole ring is key for its biological activity, and this transformation is performed by the flavin dependent halogenase MalA. This enzyme utilizes a proposed chloramine lysine intermediate to iteratively and selectively chlorinate its natural substrate premalbrancheamide. Halogenases can provide orthogonal selectivity to many chemical methods, making them useful for pharmaceutical applications, while providing selective methods for late-stage functionalization. This investigation focuses on the substrate scope of the halogenase on complex pharmaceutically relevant substrates in collaboration with the Novartis Institutes for Biomedical Research. The bromination and chlorination reaction conditions were optimized, and the products were structurally characterized by NMR spectroscopy to gain further understanding of the versatility of the wild type enzyme and its mutants

Automated genome mining for natural products

Abstract Background Discovery of new medicinal agents from natural sources has largely been an adventitious process based on screening of plant and microbial extracts combined with bioassay-guided identification and natural product structure elucidation. Increasingly rapid and more cost-effective genome sequencing technologies coupled with advanced computational power have converged to transform this trend toward a more rational and predictive pursuit. Results We have developed a rapid method of scanning genome sequences for multiple polyketide, nonribosomal peptide, and mixed combination natural products with output in a text format that can be readily converted to two and three dimensional structures using conventional software. Our open-source and web-based program can assemble various small molecules composed of twenty standard amino acids and twenty two other chain-elongation intermediates used in nonribosomal peptide systems, and four acyl-CoA extender units incorporated into polyketides by reading a hidden Markov model of DNA. This process evaluates and selects the substrate specificities along the assembly line of nonribosomal synthetases and modular polyketide synthases. Conclusion Using this approach we have predicted the structures of natural products from a diverse range of bacteria based on a limited number of signature sequences. In accelerating direct DNA to metabolomic analysis, this method bridges the interface between chemists and biologists and enables rapid scanning for compounds with potential therapeutic value.http://deepblue.lib.umich.edu/bitstream/2027.42/112362/1/12859_2008_Article_2915.pd

Identification and analysis of the bacterial endosymbiont specialized for production of the chemotherapeutic natural product ET‐743

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/115956/1/emi12908.pd