Jon D. Stewart

University of Florida, Gainesville, Florida, United States

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Publications (88)466.45 Total impact

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    ABSTRACT: Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate.
    Biomacromolecules 07/2015; DOI:10.1021/acs.biomac.5b00623 · 5.75 Impact Factor
  • Advanced Synthesis & Catalysis 05/2015; 357(8). DOI:10.1002/adsc.201500206 · 5.54 Impact Factor
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    ABSTRACT: Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase). After each round of mutagenesis, iodine staining and antibody capture activity assays at varying temperatures were used to select the optimum positions and amino acid changes for the next rounds of mutagenesis. After three iterations, the signals from whole-colony iodine staining were saturated and a heat stable AGPase variant was obtained. Kinetic studies of the heat stable mutant showed that it also had an unexpected increased affinity for the activator, 3-PGA. This is particularly valuable as both the temperature stability and allosteric properties of AGPase significantly influence grain yield.
    Archives of Biochemistry and Biophysics 01/2015; 568. DOI:10.1016/j.abb.2015.01.008 · 3.04 Impact Factor
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    ABSTRACT: An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes. Several substrates were sterically demanding, and as hoped, creating additional active site volume yielded better conversions for these alkenes. The most impressive improvement was found for 2-butylidenecyclohexanone. The wild-type provided less than 20% conversion after 24h; a triple mutant afforded more than 60% conversion in the same time period. Moreover, even wild-type OYE 2.6 can reduce cyclohexenones with very bulky 4-substituents efficiently.
    Bioorganic & Medicinal Chemistry 10/2014; 22(20). DOI:10.1016/j.bmc.2014.07.001 · 2.95 Impact Factor
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    ABSTRACT: A systematic saturation mutagenesis campaign was carried out on an alkene reductase from Pichia stipitis (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis–Hillman adducts to the corresponding S products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high R selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee (R)). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations.
    ACS Catalysis 06/2014; 4(7):2307–2318. DOI:10.1021/cs500429k · 7.57 Impact Factor
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    ABSTRACT: This study was designed to determine whether whole cells or crude enzyme extracts are more effective for preparative-scale ketone reductions by dehydrogenases as well as learning which cofactor regeneration scheme is most effective. Based on results from three representative ketone substrates (an α-fluoro-β-keto ester, a bis-trifluoromethylated acetophenone, and a symmetrical β-diketone), our results demonstrate that several nicotinamide cofactor regeneration strategies can be applied to preparative-scale dehydrogenase-catalyzed reactions successfully.
    Organic Process Research & Development 03/2014; 18(6):793–800. DOI:10.1021/op400312n · 2.55 Impact Factor
  • Sarah E Franz · Jon D Stewart
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    ABSTRACT: Threonine aldolases catalyze the pyridoxal phosphate-dependent condensation between small amino acids (principally glycine) and aldehydes such as acetaldehyde. Carbon-carbon bond formation involves forming two adjacent chiral centers. As a rule, threonine aldolases are very stereoselective for α-carbon configuration but show modest selectivity at the β-carbon. On the other hand, these enzymes accept a wide variety of synthetically useful acceptor aldehydes, making them important additions to the synthetic toolkit. This review briefly summarizes the reaction mechanism and then lists all published synthetic reactions by threonine aldolases as of early 2014. The current state of the art in crystallographic and protein engineering studies of these enzymes is also presented.
    Advances in applied microbiology 01/2014; 88:57-101. DOI:10.1016/B978-0-12-800260-5.00003-6 · 2.24 Impact Factor
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    Yuri A Pompeu · Bradford Sullivan · Jon D Stewart
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    ABSTRACT: Reductions of (S)- and (R)-carvone by wild-type Saccharomyces pastorianus Old Yellow Enzyme (OYE 1) and a systematic collection of Trp 116 variants revealed that, for (S)-carvone, six Tip 116 mutants displayed inverted diastereoselectivity compared to the wild-type. For example, Ile and Val showed inverted stereoselectivity, but Leu and Phe maintained the wild-type stereopreference. For (R)-carvone, only two Tip 116 mutants (Ala and Val) reduced this alkene with reversed selectivity; all other catalytically active variants including Leu and Ile retained the wild-type diastereoselectivity. The same set of mutant enzymes was also used to catalyze the dehydrogenation of (S)- and (R)-carvone under aerobic conditions. To understand how small changes to the active site structure of OYE 1 could significantly influence its catalytic properties, we solved X-ray crystal structures of the wildtype as well as six key Tip 116,variants after individually soaking with both (S)- and (R)-carvone. In many cases, pseudo Michaelis complexes farmed in crystallo, and these revealed the details of protein-substrate interactions. Taken together, our results showed that the wild-type OYE l'reduces carvone from a less preferred substrate binding orientation. The indole ring of Tip 116 physically blocks access to a hydrophobic active site pocket Relieving the steric congestion by mutating Tip 116 allows entry of the isopropenyl side chain of carvone into this hydrophobic pocket and also Makes the opposite face of the pi system accessible to hydride addition, thereby yielding the opposite diastereomer after net trans addition of H-2.
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    ABSTRACT: ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.
    Archives of Biochemistry and Biophysics 07/2013; DOI:10.1016/j.abb.2013.07.019 · 3.04 Impact Factor
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    Bradford Sullivan · Adam Z Walton · Jon D Stewart
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    ABSTRACT: We developed a method for creating and evaluating site-saturation libraries that consistently yields an average of 27.4±3.0 codons of the 32 possible within a pool of 95 transformants. This was verified by sequencing 95 members from 11 independent libraries within the gene encoding alkene reductase OYE 2.6 from Pichia stipitis. Correct PCR primer design as well as a variety of factors that increase transformation efficiency were critical contributors to the method's overall success. We also developed a quantitative analysis of library quality (Q-values) that defines library degeneracy. Q-values can be calculated from standard fluorescence sequencing data (capillary electropherograms) and the degeneracy predicted from an early stage of library construction (pooled plasmids from the initial transformation) closely matched that observed after ca. 1000 library members were sequenced. Based on this experience, we suggest that this analysis can be a useful guide when applying our optimized protocol to new systems, allowing one to focus only on good-quality libraries and reject substandard libraries at an early stage. This advantage is particularly important when lower-throughput screening techniques such as chiral-phase GC must be employed to identify protein variants with desirable properties, e.g., altered stereoselectivities or when multiple codons are targeted for simultaneous randomization.
    06/2013; 53(1):70-77. DOI:10.1016/j.enzmictec.2013.02.012
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    ABSTRACT: ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes.With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last.3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms.By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes.Redox state does not affect kcat of the two potato isoforms.Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex.This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme.Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis.
    Archives of Biochemistry and Biophysics 04/2013; 535(2). DOI:10.1016/j.abb.2013.04.003 · 3.04 Impact Factor
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    ABSTRACT: Recent data suggest alternative mechanisms that promote human leukocyte antigen (HLA)-associated drug syndromes. Hypersensitive responses have been attributed to drug interactions with HLA molecules, peptides presented by HLA molecules and T-cell antigen receptors. Definition of an increasing number of HLA-associated drug syndromes suggests that polymorphism in the antigen-binding cleft residues influence recognition of specific drugs. Recent data demonstrate that small molecule drugs bind within the antigen-binding cleft of HLA in a manner that alters the repertoire of HLA-bound peptide ligands. This drug recognition mechanism permits presentation of self-peptides to which the host has not been tolerized. This altered repertoire mechanism is analogous to massive polyclonal T-cell responses occurring in mismatched HLA organ transplantation in which the drug in effect creates a novel HLA allele. Alteration of the self-peptide repertoire by HLA-binding small molecules may be the mechanistic basis for a diverse set of deleterious T-cell responses since the antigen-binding cleft has structural features that are compatible with binding drug-like small molecules. Small molecule drugs that bind elements of the trimolecular complex (T-cell receptor, peptide, and HLA) may cause short- and long-term adverse effects by a diverse set of mechanisms.
    Immunological Reviews 11/2012; 250(1):158-66. DOI:10.1111/j.1600-065X.2012.01163.x · 12.91 Impact Factor
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    ABSTRACT: We have probed Pichia stipitis CBS 6054 Old Yellow Enzyme 2.6 (OYE 2.6) by several strategies including X-ray crystallography, ligand binding and catalytic assays using the wild-type as well as libraries of site-saturation mutants. The alkene reductase crystallized in space group P?63?2?2 with unit cell dimensions of 127.1x123.4 angstrom and its structure was solved to 1.5 angstrom resolution by molecular replacement. The protein environment surrounding the flavin mononucleotide (FMN) cofactor was very similar to those of other OYE superfamily members; however, differences in the putative substrate binding site were also observed. Substrate analog complexes were analyzed by both UV-Vis titration and X-ray crystallography to provide information on possible substrate binding interactions. In addition, four active site residues were targeted for site saturation mutagenesis (Thr 35, Ile 113, His 188, His 191) and each library was tested against three representative BaylisHillman adducts. Thr 35 could be replaced by Ser with no change in activity; other amino acids (Ala, Cys, Leu, Met, Gln and Val) resulted in diminished catalytic efficiency. The Ile 113 replacement library yielded a range of catalytic activities, but had very little impact on stereoselectivity. Finally, the two His residues (188 and 191) were essentially intolerant of substitutions with the exception of the His 191 Asn mutant, which did show significant catalytic ability. Structural comparisons between OYE 2.6 and Saccharomyces pastorianus OYE1 suggest that the key interactions between the substrate hydroxymethyl groups and the side-chain of Thr 35 and/or Tyr 78 play an important role in making OYE 2.6 an (S)-selective alkene reductase.
    Advanced Synthesis & Catalysis 07/2012; 354(10-10):1949-1960. DOI:10.1002/adsc.201200213 · 5.54 Impact Factor
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    ABSTRACT: The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADP-glucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33 °C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only ∼50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.
    The Plant Cell 06/2012; 24(6):2352-63. DOI:10.1105/tpc.112.100602 · 9.58 Impact Factor
  • Despina J. Bougioukou · Jon D. Stewart
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    ABSTRACT: This chapter contains sections titled: IntroductionAlkene Reduction by Whole Microbial CellsAlkene Reductions by Isolated EnzymesApplications of Alkene ReductasesAccessing Both Product EnantiomersReferences
    Enzyme Catalysis in Organic Synthesis, 04/2012: pages 1111-1163; , ISBN: 9783527325474
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    ABSTRACT: Baylis-Hillman adducts are highly useful synthetic intermediates; to enhance their value further, we sought enantiocomplementary alkene reductases to introduce chirality. Two solutions emerged: (1) a wild-type protein from Pichia stipitis (OYE 2.6), whose performance significantly outstrips that of the standard enzyme (Saccharomyces pastorianus OYE1), and (2) a series of OYE1 mutants at position 116 (Trp in the wild-type enzyme). To understand how mutations could lead to inverted enantioselectivity, we solved the X-ray crystal structure of the Trp116Ile OYE1 variant complexed with a cyclopentenone substrate. This revealed key protein-ligand interactions that control the orientation of substrate binding above the FMN cofactor.
    ACS Catalysis 09/2011; 1(9). DOI:10.1021/cs200223f · 7.57 Impact Factor
  • Jillian L Perry · Charles R Martin · Jon D Stewart
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    ABSTRACT: Encapsulating drugs within hollow nanotubes offers several advantages, including protection from degradation, the possibility of targeting desired locations, and drug release only under specific conditions. Template synthesis utilizes porous membranes prepared from alumina, polycarbonate, or other materials that can be dissolved under specific conditions. The method allows for great control over the lengths and diameters of nanotubes; moreover, tubes can be constructed from a wide variety of tube materials including proteins, DNA, silica, carbon, and chitosan. A number of capping strategies have been developed to seal payloads within nanotubes. Combining these advances with the ability to target and internalize nanotubes into living cells will allow these assemblies to move into the next phase of development, in vivo experiments.
    Chemistry - A European Journal 05/2011; 17(23):6296-302. DOI:10.1002/chem.201002835 · 5.70 Impact Factor
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    ABSTRACT: Recent publications have suggested that cylindrically shaped drug-delivery carriers have an advantage over carriers based on spherical particles in both blood circulation and cell internalization rates. For this reason, this article introduces a method to fabricate hollow, uniform, biodegradable chitosan nano test tubes for applications in drug delivery. A nanoporous alumina template membrane was used to fabricate hollow chitosan nano test tubes. The chitosan nano test tubes were crosslinked with a disulfide cleavable crosslinker before being removed from the alumina template membrane. We explored two mechanisms for degrading the chitosan nano test tubes--enzymatic degradation by lysozyme and cleavage of the disulfide bond in the crosslinking agent. The template synthesis method resulted in the fabrication of uniform hollow chitosan nano test tubes whose dimensions were easily manipulated based on the dimensions of the pores in the alumina template membrane. The tubes were degraded upon exposure to either lysozyme or sulfhydryl-containing reducing reagents. These tubes have potential for drug-delivery applications. The fact that these tubes degrade upon exposure to a sulfhydryl-containing reducing agent allows for a mechanism for intercellular drug delivery as the tubes should degrade in the presence of intercellular glutathione.
    Nanomedicine 10/2010; 5(8):1151-60. DOI:10.2217/nnm.10.110 · 5.82 Impact Factor
  • Despina J Bougioukou · Adam Z Walton · Jon D Stewart
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    ABSTRACT: Simple strategies for using alkene reductase enzymes to produce gram-scale quantities of both (R)- and (S)-citronellal have been developed. The methodology is easily accessible to non-specialist laboratories, allowing alkene reductases to be added to the toolbox of routine synthetic transformations.
    Chemical Communications 10/2010; 46(45):8558-60. DOI:10.1039/c0cc03119d · 6.83 Impact Factor
  • ChemInform 08/2010; 31(32). DOI:10.1002/chin.200032064

Publication Stats

2k Citations
466.45 Total Impact Points

Institutions

  • 1996–2015
    • University of Florida
      • • Department of Chemistry
      • • Department of Biomedical Engineering
      Gainesville, Florida, United States
  • 2010
    • Vienna University of Technology
      • Institute of Applied Synthetic Chemistry
      Wien, Vienna, Austria
  • 1998–2001
    • University of New Brunswick
      • Department of Chemistry
      Fredericton, New Brunswick, Canada
  • 1999
    • Hokkaido University
      • Graduate School of Pharmaceutical Sciences
      Sapporo-shi, Hokkaido, Japan
  • 1995
    • Johns Hopkins University
      • Department of Biophysics
      Baltimore, Maryland, United States
  • 1994
    • The Scripps Research Institute
      لا هویا, California, United States
  • 1993
    • Pennsylvania State University
      • Department of Chemistry
      University Park, Maryland, United States