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Altering Oligomerization Of Epha2 Via Mutations In The Intracellular Domain, Ryan W. Lingerak 2018 The University of Akron

Altering Oligomerization Of Epha2 Via Mutations In The Intracellular Domain, Ryan W. Lingerak

Honors Research Projects

Eph receptor tyrosine kinases (RTKs) are activated by membrane-bound ligands called ephrins. Eph RTKs are divided into two subclasses, each activated by a specific classes of the ligand ephrin. The overexpression of Eph receptors is correlated to cancer cell metastasis in several different types of cancers. Studies with the EphA2 extracellular domain (ECD) and ephrinA1 ligand have shown that upon binding of ephrin to the receptor, EphA2 undergoes increased oligomerization and activation. This indicates that oligomerization is intimately connected to kinase activity. High resolution crystal structures of the EphA2 ECD have revealed some details of these ligand bound oligomers, as ...


A Single Residue Switch Converts Abietadiene Synthase Into A Pimaradiene Specific Cyclase, P. Ross Wilderman, Reuben J. Peters 2017 Iowa State University

A Single Residue Switch Converts Abietadiene Synthase Into A Pimaradiene Specific Cyclase, P. Ross Wilderman, Reuben J. Peters

Reuben J. Peters

Terpene synthases often catalyze complex cyclization reactions that typically represent the committed step in particular biosynthetic pathways, leading to great interest in their enzymatic mechanisms. We have recently demonstrated that substitution of a specific Ile with Thr was sufficient to “short circuit” the complex cyclization reaction normally catalyzed by ent-kaurene synthases to instead produce ent-pimaradiene. Here we report that the complex cyclization/rearrangement reaction catalyzed by abietadiene synthase can be similarly cut short to produce pimaradienes by an analogous Ser for Ala change, albeit with a slight shift in active site location to accommodate the difference in substrate ...


A Functional Genomics Approach To Tanshinone Biosynthesis Provides Stereochemical Insights, Wei Gao, Matthew L. Hillwig, Luqi Huang, Guanghong Cui, Xueyong Wang, Jianqiang Kong, Bin Yang, Reuben J. Peters 2017 Chinese Academy of Chinese Medical Science

A Functional Genomics Approach To Tanshinone Biosynthesis Provides Stereochemical Insights, Wei Gao, Matthew L. Hillwig, Luqi Huang, Guanghong Cui, Xueyong Wang, Jianqiang Kong, Bin Yang, Reuben J. Peters

Reuben J. Peters

Tanshinones are abietane-type norditerpenoid quinone natural products that are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza Bunge. The initial results from a functional genomics-based investigation of tanshinone biosynthesis, specifically the functional identification of the relevant diterpene synthases from S. miltiorrhiza, are reported. The cyclohexa-1,4-diene arrangement of the distal ring poises the resulting miltiradiene for the ensuing aromatization and hydroxylation to ferruginol suggested for tanshinone biosynthesis.


Interdependence Of Inhibitor Recognition In Hiv-1 Protease, Janet L. Paulsen, Florian Leidner, Debra A. Ragland, Nese Kurt Yilmaz, Celia A. Schiffer 2017 University of Massachusetts Medical School

Interdependence Of Inhibitor Recognition In Hiv-1 Protease, Janet L. Paulsen, Florian Leidner, Debra A. Ragland, Nese Kurt Yilmaz, Celia A. Schiffer

Celia A. Schiffer

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency ...


An Approach To Identify Mycobacteriophage Diversity Prior To Dna Sequencing, Charles Gregory 2017 Western Kentucky University

An Approach To Identify Mycobacteriophage Diversity Prior To Dna Sequencing, Charles Gregory

Honors College Capstone Experience/Thesis Projects

Over 6,869 Mycobacteriophages have been isolated and purified. Of these, 1,367 genomes have been sequenced at the DNA level and more are added each year through the SEA-PHAGES program. Sequenced mycobacteriophages are grouped into clusters based on a 50% or greater nucleotide identity. The number and breadth of these clusters represents the diversity present in the environment. Each year, as new phages are discovered by students in the SEA-PHAGES program, the question arises, “Which isolates should we sequence?” In order to sequence phages that represent the greatest possible diversity, and thus broaden under-represented clusters and identify new singletons ...


Edaxadiene: A New Bioactive Diterpene From Mycobacterium Tuberculosis, Francis M. Mann, Meimei Xu, Xiaoming Chen, D. Bruce Fulton, David G. Russell, Reuben J. Peters 2017 Iowa State University

Edaxadiene: A New Bioactive Diterpene From Mycobacterium Tuberculosis, Francis M. Mann, Meimei Xu, Xiaoming Chen, D. Bruce Fulton, David G. Russell, Reuben J. Peters

Reuben J. Peters

Mycobacterium tuberculosis remains a widespread and devastating human pathogen. Presented here is the characterization of an atypical class I diterpene cyclase from M. tuberculosis that catalyzes an unusual cyclization reaction in converting the known M. tuberculosis metabolite halimadienyl diphosphate to a further cyclized novel diterpene, which we have termed edaxadiene, as it directly inhibits maturation of the phagosomal compartment in which the bacterium is taken up during infection.


Insights Into Diterpene Cyclization From Structure Of Bifunctional Abietadiene Synthase From Abies Grandis, Ke Zhou, Yang Gao, Julie A. Hoy, Francis M. Mann, Richard B. Honzatko, Reuben J. Peters 2017 Iowa State University

Insights Into Diterpene Cyclization From Structure Of Bifunctional Abietadiene Synthase From Abies Grandis, Ke Zhou, Yang Gao, Julie A. Hoy, Francis M. Mann, Richard B. Honzatko, Reuben J. Peters

Reuben J. Peters

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of ...


Isotuberculosinol: The Unusual Case Of An Immunomodulatory Diterpenoid From Mycobacterium Tuberculosis, Francis M. Mann, Reuben J. Peters 2017 Winona State University

Isotuberculosinol: The Unusual Case Of An Immunomodulatory Diterpenoid From Mycobacterium Tuberculosis, Francis M. Mann, Reuben J. Peters

Reuben J. Peters

The actinomycetes family of bacteria is classically considered a rich source of natural products [1]. It is often suggested that such compounds have been selected by evolution to have biological activity [2]. For example, given the microbial-rich soil environment of the actinomycetes native habitat, their natural products have been suggested to serve in chemical warfare and/or signaling, which has been further connected to their leading role in providing pharmaceutically relevant antibiotic agents [3]. Nevertheless, surprisingly few of these molecules have been definitively assigned specific physiological roles in the native producing bacteria.


Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson 2017 University of Pennsylvania

Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson

Reuben J. Peters

The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.


Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters 2017 Iowa State University

Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters

Reuben J. Peters

In his original exposition of the biogenetic isoprenoid rule, L. Ruzicka noted the structural identity between the fused A/B rings of triterpenoids/sterols and certain multicyclic diterpenoids as part of the impetus leading to that profound insight. His prescient hypothesis that this chemical structure relationship reflects similarities in the initial cyclization of these diterpenoids with that occurring in triterpenoid biosynthesis has since been verified. However, this chemical structure relationship does not continue to hold true for the additional rings found in many of these di- and tri- terpenoid natural products. This is now appreciated to arise from differences in ...


Terpenoid Synthase Structures: A So Far Incomplete View Of Complex Catalysis, Yang Gao, Richard B. Honzatko, Reuben J. Peters 2017 Iowa State University

Terpenoid Synthase Structures: A So Far Incomplete View Of Complex Catalysis, Yang Gao, Richard B. Honzatko, Reuben J. Peters

Reuben J. Peters

The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the ...


Functional Characterization Of Wheat Copalyl Diphosphate Synthases Sheds Light On The Early Evolution Of Labdane-Related Diterpenoid Metabolism In The Cereals, Yisheng Wu, Ke Zhou, Tomonobu Toyomasu, Chizu Sugawara, Madoka Oku, Shiho Abe, Masami Usui, Wataru Mitsuhashi, Makiko Chono, Peter M. Chandler, Reuben J. Peters 2017 Iowa State University

Functional Characterization Of Wheat Copalyl Diphosphate Synthases Sheds Light On The Early Evolution Of Labdane-Related Diterpenoid Metabolism In The Cereals, Yisheng Wu, Ke Zhou, Tomonobu Toyomasu, Chizu Sugawara, Madoka Oku, Shiho Abe, Masami Usui, Wataru Mitsuhashi, Makiko Chono, Peter M. Chandler, Reuben J. Peters

Reuben J. Peters

Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals – specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1,2,&3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then revealed that TaCPS2 uniquely produces normal, rather than ent-, CPP; thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP ...


A Single Residue Switch For Mg2+-Dependent Inhibition Characterizes Plant Class Ii Diterpene Cyclases From Primary And Secondary Metabolism, Francis M. Mann, Sladjana Prisic, Emily K. Davenport, Mara K. Determan, Robert M. Coates, Reuben J. Peters 2017 Iowa State University

A Single Residue Switch For Mg2+-Dependent Inhibition Characterizes Plant Class Ii Diterpene Cyclases From Primary And Secondary Metabolism, Francis M. Mann, Sladjana Prisic, Emily K. Davenport, Mara K. Determan, Robert M. Coates, Reuben J. Peters

Reuben J. Peters

Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg2+ cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg2+ binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated ...


Diterpenoid Biopolymers: New Directions For Renewable Materials Engineering, Matthew L. Hillwig, Francis M. Mann, Reuben J. Peters 2017 Iowa State University

Diterpenoid Biopolymers: New Directions For Renewable Materials Engineering, Matthew L. Hillwig, Francis M. Mann, Reuben J. Peters

Reuben J. Peters

Most types of ambers are naturally occurring, relatively hard, durable resinite polymers derived from the exudates of trees. This resource has been coveted for thousands of years due to its numerous useful properties in industrial processes, beauty, and purported medicinal properties. Labdane diterpenoid based ambers represent the most abundant and important resinites on earth. These resinites are a dwindling, non-renewable natural resource, so a new source of such materials needs to be established. Recent advances in sequencing technologies and biochemical engineering are rapidly accelerating the rate of identifying and assigning function to genes involved in terpenoid biosynthesis, as well as ...


Electrostatic Effects On (Di)Terpene Synthase Product Outcome, Ke Zhou, Reuben J. Peters 2017 Iowa State University

Electrostatic Effects On (Di)Terpene Synthase Product Outcome, Ke Zhou, Reuben J. Peters

Reuben J. Peters

Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to exert little control over the actual reaction, instead largely serving as inert templates. However, recent results highlight stereoelectronic effects exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may drive carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues ...


A Novel Labda-7,13e-Dien-15-Ol-Producing Bifunctional Diterpene Synthase From Selaginella Moellendorffii, Sibongile Mafu, Matthew L. Hillwig, Reuben J. Peters 2017 Iowa State University

A Novel Labda-7,13e-Dien-15-Ol-Producing Bifunctional Diterpene Synthase From Selaginella Moellendorffii, Sibongile Mafu, Matthew L. Hillwig, Reuben J. Peters

Reuben J. Peters

Vascular plants invariably contain a class II diterpene cyclase (EC 5.5.1.x), as an ent-copalyl diphosphate synthase is required for gibberellin phytohormone biosynthesis. This has provided the basis for evolution of a functionally diverse enzymatic family.[1] These biocatalysts fold their substrate, the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP), to bring the terminal three carbon-carbon double bonds into proximity with each other, and then carry out bicyclization via a protonation-initiated carbocation cascade reaction. The resulting labda-15-en-8-yl+ diphosphate intermediate is most commonly quenched by deprotonation at an exocyclic methyl, as in the production of labdadienyl/copalyl ...


Evident And Latent Plasticity Across The Rice Diterpene Synthase Family With Potential Implications For The Evolution Of Diterpenoid Metabolism In The Cereals, Dana Morrone, Matthew L. Hillwig, Matthew E. Mead, Luke Lowry, D. Bruce Fulton, Reuben J. Peters 2017 Iowa State University

Evident And Latent Plasticity Across The Rice Diterpene Synthase Family With Potential Implications For The Evolution Of Diterpenoid Metabolism In The Cereals, Dana Morrone, Matthew L. Hillwig, Matthew E. Mead, Luke Lowry, D. Bruce Fulton, Reuben J. Peters

Reuben J. Peters

The evolution of natural products biosynthetic pathways can be envisioned to occur via a number of mechanisms. Here we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-copalyl diphosphate (CPP), which are substrates for downstream diterpene synthases. Here we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent-, syn-, or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported entsandaracopimaradiene synthase also converts syn-CPP to syn-labda-8(17),12E,14-triene, which can be found in planta. Notably, normal CPP ...


Picking Sides: Distinct Roles For Cyp76m6 And Cyp76m8 In Rice Oryzalexin Biosynthesis, Yisheng Wu, Qiang Wang, Matthew L. Hillwig, Reuben J. Peters 2017 Iowa State University

Picking Sides: Distinct Roles For Cyp76m6 And Cyp76m8 In Rice Oryzalexin Biosynthesis, Yisheng Wu, Qiang Wang, Matthew L. Hillwig, Reuben J. Peters

Reuben J. Peters

Natural products biosynthesis often requires the action of multiple cytochromes P450 (CYPs), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we have characterized CYPs that catalyze alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, -6 and -8 were all shown to carry out C7β-hydroxylation, while CYP701A8 catalyzes C3α-hydroxylation, with oxy groups found at both positions in oryzalexins A–D, suggesting that these may act consecutively in oryzalexin biosynthesis. Here we report that, although CYP701A8 only poorly reacts with 7β-hydroxy-entsandaracopimaradiene, CYP76M6 and ...


Product Rearrangement From Altering A Single Residue In The Rice Syn-Copalyl Diphosphate Synthase, Kevin C. Potter, Meirong Jia, Young J. Hong, Dean Tantillo, Reuben J. Peters 2017 Iowa State University

Product Rearrangement From Altering A Single Residue In The Rice Syn-Copalyl Diphosphate Synthase, Kevin C. Potter, Meirong Jia, Young J. Hong, Dean Tantillo, Reuben J. Peters

Reuben J. Peters

Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism.


Probing Labdane-Related Diterpenoid Biosynthesis In The Fungal Genus Aspergillus, Meimei Xu, Matthew L. Hillwig, Mollie S. Tiernan, Reuben J. Peters 2017 Iowa State University

Probing Labdane-Related Diterpenoid Biosynthesis In The Fungal Genus Aspergillus, Meimei Xu, Matthew L. Hillwig, Mollie S. Tiernan, Reuben J. Peters

Reuben J. Peters

While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid ...


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