Recycling Upstream Redox Enzymes Expands the Regioselectivity of Cycloaddition in Pseudo-Aspidosperma Alkaloid Biosynthesis

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudoaspidosperma alkaloids pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors

Hardware Validation Test of the Advanced Plant Habitat

An automated plant growth facility for conducting plant research supporting space biology and food production project on the International Space Station (ISS)

Structure and Biochemistry of Cytochromes P450 Involved in the Biosynthesis of Macrolide Antibiotics

Selective functionalization of chemically inert carbon-hydrogen (C–H) bonds embodies one of the grand challenges of organic chemistry and provides a key focus of research in the field. C–H functionalization can provide a valuable means to improve the efficiency of complex molecule synthesis, but significant challenges remain with respect to achieving high site selectivity in the presence of multiple unique C–H bonds in a given target. As a complement to small-molecule transition metal-based catalysts, enzymes have received increasing attention in recent years as potential biocatalysts for carrying out selective C–H bond oxidation reactions. Members of the cytochrome P450 superfamily of monooxygenases (P450s) are some of nature’s most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize C–H bonds has led to their growing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. The work presented herein details studies that were carried out to biochemically and structurally characterize diverse bacterial P450s involved in the biosynthesis of 16-membered ring macrolide antibiotics with significant potential for development into robust biocatalysts for the late-stage functionalization of complex molecules. In an initial study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding and kinetics experiments along with X-ray crystallographic analysis of MycCI bound to its native substrate. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared with that of MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity. Next, we performed a more in-depth analysis of TylHI in an attempt to understand the molecular basis for its substrate specificity. Turnover and equilibrium binding experiments with substrate analogs revealed that this enzyme exhibits a strict preference for 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. These results were partially explained through analysis of the X-ray crystal structure of TylHI in complex with its native substrate together with biochemical characterization of several site-directed mutants. Comparative analysis of the MycCI/TylHI homolog ChmHI from the chalcomycin biosynthetic pathway provided a basis for constructing MycCI/TylHI chimeras in order to gain further insight into the features dictating the differences in the reactivity profiles of these two related P450s. These experiments unveiled the central role of the BC loop region in influencing the binding properties of 16-membered ring substrates to MycCI and TylHI. Finally, comparative analysis of several different P450s from the mycinamicin (MycCI), tylosin (TylI), and juvenimicin (JuvC and JuvD) biosynthetic pathways revealed unique substrate preferences and catalytic outcomes that facilitated their subsequent employment as biocatalysts in the chemoenzymatic synthesis of tylactone-based macrolide antibiotics.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140964/1/mdemars_1.pd

Providing Centralized Access to CAS 352 Research Projects

paper, CAS352/BIO, spring 201

Moose, caribou and fire: have we got it right yet?

Natural disturbance plays a key role in shaping community dynamics. Within Canadian boreal forests, the dominant form of natural disturbance is fire, and its effects are thought to influence the dynamics between moose (Alces alces (Linnaeus, 1758)) and the boreal ecotype of woodland caribou (Rangifer tarandus caribou (Gmelin, 1788)). Boreal caribou are considered “threatened” and population declines are attributed, at least in part, to disturbance-mediated apparent competition (DMAC) with moose. Here, we tested a primary prediction of the DMAC hypothesis: that moose respond positively to burns within and adjacent to the caribou range. We assessed moose selection for ≤25-year-old burns (when selection is predicted to be strongest) at multiple spatial scales and evaluated whether moose density was correlated with the extent of ≤40-year-old burns (a time frame predicted to negatively affect caribou). Against expectation, moose showed avoidance and low use of ≤25-year-old burns at all scales, regardless of burn age, season, and type of land cover burned. These findings mirrored the demographic response, as we found no correlation between ≤40-year-old burns and moose density. By contradicting the prevailing hypothesis linking fires to caribou population declines, our results highlight the need to understand regional variation in disturbance impacts on caribou populations.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Computational-Based Mechanistic Study and Engineering of Cytochrome P450 MycG for Selective Oxidation of 16-Membered Macrolide Antibiotics

MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide M-IV. The enzyme evolved to oxidize M-IV preferentially over M-III and M-VI, which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG. Density functional theory computations determined that epoxidation of the substrate hampers its ability to undergo C-H abstraction, primarily due to a loss of hyperconjugation in the transition state. Metadynamics and molecular dynamics simulations revealed a hydrophobic sugar-binding pocket that is responsible for substrate recognition/specificity and was not apparent in crystal structures of the enzyme/substrate complex. Computational results also led to the identification of other interactions between the enzyme and its substrates that had not previously been observed in the cocrystal structures. Site-directed mutagenesis was then employed to test the effects of mutations hypothesized to broaden the substrate scope and alter the product profile of MycG. The results of these experiments validated this complementary effort to engineer MycG variants with improved catalytic activity toward earlier stage mycinamicin substrates

Controlled Oxidation of Remote sp³ C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity

The selective oxyfunctionalization of isolated sp³ C–H bonds in complex molecules represents a formidable challenge in organic chemistry. Here, we describe a rational, systematic strategy to expedite the development of P450 oxidation catalysts with refined regio- and stereoselectivity for the hydroxylation of remote, unactivated C–H sites in a complex scaffold. Using artemisinin as model substrate, we demonstrate how a three-tier strategy involving first-sphere active site mutagenesis, high-throughput P450 fingerprinting, and fingerprint-driven P450 reactivity predictions enabled the rapid evolution of three efficient biocatalysts for the selective hydroxylation of a primary and a secondary C–H site (with both <i>S</i> and <i>R</i> stereoselectivity) in a relevant yet previously inaccessible region of this complex natural product. The evolved P450 variants could be applied to provide direct access to the desired hydroxylated derivatives at preparative scales (0.4 g) and in high isolated yields (>90%), thereby enabling further elaboration of this molecule. As an example, enantiopure C7-fluorinated derivatives of the clinical antimalarial drugs artesunate and artemether, in which a major metabolically sensitive site is protected by means of a C–H to C–F substitution, were afforded via P450-mediated chemoenzymatic synthesis

Exploring the molecular basis for substrate specificity in homologous macrolide biosynthetic cytochromes P450

Cytochromes P450 (P450s) are nature's catalysts of choice for performing demanding and physiologically vital oxidation reactions. Biochemical characterization of these enzymes over the past decades has provided detailed mechanistic insight and highlighted the diversity of substrates P450s accommodate and the spectrum of oxidative transformations they catalyze. Previously, we discovered that the bacterial P450 MycCI from the mycinamicin biosynthetic pathway in Micromonospora griseorubida possesses an unusually broad substrate scope, whereas the homologous P450 from tylosin-producing Streptomyces fradiae (TylHI) exhibits a high degree of specificity for its native substrate. Here, using biochemical, structural, and computational approaches, we aimed to understand the molecular basis for the disparate reactivity profiles of these two P450s. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI strictly prefers 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. To help rationalize these results, we solved the X-ray crystal structure of TylHI in complex with its native substrate at 1.99-Å resolution and assayed several site-directed mutants. We also conducted molecular dynamics simulations of TylHI and MycCI and biochemically characterized a third P450 homolog from the chalcomycin biosynthetic pathway in Streptomyces bikiniensis These studies provided a basis for constructing P450 chimeras to gain further insight into the features dictating the differences in reaction profile among these structurally and functionally related enzymes, ultimately unveiling the central roles of key loop regions in influencing substrate binding and turnover. Our work highlights the complex nature of P450/substrate interactions and raises interesting questions regarding the evolution of functional diversity among biosynthetic enzymes