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

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

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

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

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

Comparative genomic hybridizations reveal absence of large Streptomyces coelicolor genomic islands in Streptomyces lividans

<p>Abstract</p> <p>Background</p> <p>The genomes of <it>Streptomyces coelicolor </it>and <it>Streptomyces lividans </it>bear a considerable degree of synteny. While <it>S. coelicolor </it>is the model streptomycete for studying antibiotic synthesis and differentiation, <it>S. lividans </it>is almost exclusively considered as the preferred host, among actinomycetes, for cloning and expression of exogenous DNA. We used whole genome microarrays as a comparative genomics tool for identifying the subtle differences between these two chromosomes.</p> <p>Results</p> <p>We identified five large <it>S. coelicolor </it>genomic islands (larger than 25 kb) and 18 smaller islets absent in <it>S. lividans </it>chromosome. Many of these regions show anomalous GC bias and codon usage patterns. Six of them are in close vicinity of tRNA genes while nine are flanked with near perfect repeat sequences indicating that these are probable recent evolutionary acquisitions into <it>S. coelicolor</it>. Embedded within these segments are at least four DNA methylases and two probable methyl-sensing restriction endonucleases. Comparison with <it>S. coelicolor </it>transcriptome and proteome data revealed that some of the missing genes are active during the course of growth and differentiation in <it>S. coelicolor</it>. In particular, a pair of methylmalonyl CoA mutase (<it>mcm</it>) genes involved in polyketide precursor biosynthesis, an acyl-CoA dehydrogenase implicated in timing of actinorhodin synthesis and <it>bldB</it>, a developmentally significant regulator whose mutation causes complete abrogation of antibiotic synthesis belong to this category.</p> <p>Conclusion</p> <p>Our findings provide tangible hints for elucidating the genetic basis of important phenotypic differences between these two streptomycetes. Importantly, absence of certain genes in <it>S. lividans </it>identified here could potentially explain the relative ease of DNA transformations and the conditional lack of actinorhodin synthesis in <it>S. lividans</it>.</p