Jon D Stewart

University of Florida, Gainesville, Florida, United States

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Publications (54)223.35 Total impact

  • [Show abstract] [Hide abstract]
    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. · 5.27 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. · 2.74 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. 01/2014;
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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 Trp 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 Trp 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 wild-type as well as six key Trp 116 variants after individually soaking with both (S)-and (R)-carvone. In many cases, pseudo-Michaelis complexes formed in crystallo, and these revealed the details of protein−substrate interactions. Taken together, our results showed that the wild-type OYE 1 reduces carvone from a less preferred substrate binding orientation. The indole ring of Trp 116 physically blocks access to a hydrophobic active site pocket. Relieving the steric congestion by mutating Trp 116 allows entry of the isopropenyl side-chain of carvone into this hydrophobic pocket and also makes the opposite face of the π system accessible to hydride addition, thereby yielding the opposite diastereomer after net trans-addition of H 2 . KEYWORDS: alkene reductase, ene reductase, carvone, X-ray crystallography, binding orientation, mutant ■ INTRODUCTION Enzymes of the Old Yellow Enzyme (OYE) family have recently gained favor as stereoselective catalysts for reducing electron-deficient alkenes (for recent reports, see ref 1 and references therein). Reduction proceeds by a net trans-addition of H 2 to olefins, 2 particularly those conjugated with aldehydes, ketones, nitriles, nitro groups, and, in some cases, esters. Hydrogenation follows a polar mechanism in which hydride is transferred from reduced flavin mononucleotide (FMN) to the electron-deficient β-carbon with concomitant protonation at the α-carbon. 3 Both the stereochemical course and substrate range of OYEs complement those of most organometallic hydrogenation catalysts, which has led to a renewed interest in this class of flavoproteins, originally discovered in 1932 by Warburg. 4 One challenge in developing biocatalytic methods for organic synthesis is that one enantiomer is often easily available, but the "other" enantiomer is not. 5 The very high stereoselectivities of OYEs are thus a double-edged sword that make it difficult to access both product stereoisomers unless the substrate structure can be altered, i.e., employing the opposite alkene configuration. 6 For example, wild-type Saccharomyces pastor-ianus 7 OYE 1 reduces simple α-alkyl-substituted 2-cyclo-hexenones, e.g., 2-methyl-2-cyclohexenone, to the correspond-ing (R)-products with very high stereoselectivities. 8 The same enzyme reduces both enantiomers of carvone to the analogous products (Scheme 1). 9 All of these reduction products derive from Michaelis complexes in which the 2-cyclohexenone ring can be superimposed on the aromatic ring carbons of p-hydroxybenzaldehyde, a potent OYE 1 inhibitor observed in the X-ray crystal structure of S. pastorianus OYE 1. 10 For simplicity, we refer to this arrangement as the "normal" substrate binding mode. With one exception (vide infra), we were unable to discover an enantiocomplementary OYE for any of the substrates we investigated and were therefore able to fulfill only half of our methodological goals. A more general solution to this problem is reported here that joins some additional successes in this area reported by other groups. 11 In principle, one could either invert the stereoselectivity of an OYE by relocating the substrate from one side of the FMN cofactor to the other, or by turning the substrate over, thereby exposing the opposite face of the alkene π system to the FMN. Only the si face of the FMN cofactor is accessible in the active sites of OYEs, and prospects for remodeling the protein to accommodate substrate binding on the opposite face of the cofactor appeared bleak. We therefore concentrated on strategies to invert the orientation of substrate binding with respect to the FMN cofactor (referred to as "flipped" substrate binding, Scheme 1). The first steps toward achieving this goal came from a chance observation. Like many OYE family
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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; · 3.37 Impact Factor
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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.
    Enzyme and microbial technology. 06/2013; 53(1):70-77.
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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; · 3.37 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. · 12.16 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. · 9.25 Impact Factor
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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. · 5.93 Impact Factor
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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 01/2011; · 5.27 Impact Factor
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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. · 6.38 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. · 5.26 Impact Factor
  • ChemInform 01/2010; 31(32).
  • ChemInform 01/2010; 32(27).
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 33(31).
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    ABSTRACT: ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA--with or without 5.0 mm inorganic phosphate--ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K(M) values for both substrates.
    Plant physiology 12/2009; 152(2):1056-64. · 6.56 Impact Factor
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    ABSTRACT: Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined--in both the small and large subunits--bound 3-PGA more weakly than the wild type (A(50) increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.
    Plant physiology 11/2009; 152(1):85-95. · 6.56 Impact Factor
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    ABSTRACT: Site-saturation mutagenesis was used to generate all possible replacements for Trp 116 of Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis ) old yellow enzyme (OYE). Our original hypothesisthat smaller amino acids at position 116 would allow better acceptance of bulky 3-alkyl-substituted 2-cyclohexenonesproved incorrect. Instead, Phe and Ile replacements favored the binding of some substrates in an opposite orientation, which yielded reversed stereochemical outcomes compared to that of the wild-type OYE. For example, W116I OYE reduced (R)- and (S)-carvone to enantiomeric products, rather than the diastereomers produced by the wild-type OYE. Deuterium labeling revealed that (S)-carvone reduction by the W116I OYE occurred by the same pathway as that by the wild type (net trans-addition of H(2)), proving that different substrate binding orientations were responsible for the divergent products. Trp 116 mutants also afforded different stereochemical outcomes for reductions of (R)-perillaldehyde and neral. Preliminary studies of an OYE family member whose native sequence contains Ile at position 116 ( Pichia stipitis OYE 2.6) revealed that this enzyme's stereoselectivity matched that of the wild-type S. pastorianus OYE, showing that the identity of the residue at position 116 does not solely determine the substrate binding orientation. Computational docking studies using an induced fit methodology successfully reproduced the majority of the experimental outcomes. These computational tools will allow preliminary in silico screening of additional residues to identify those most likely to control the substrate binding orientation and provide some guidance to future experimental studies.
    Journal of the American Chemical Society 03/2009; 131(9):3271-80. · 10.68 Impact Factor
  • Adam Z Walton, Jon D Stewart
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    ABSTRACT: Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.
    Biotechnology Progress 09/2008; 18(2):262-8. · 1.85 Impact Factor

Publication Stats

619 Citations
223.35 Total Impact Points

Institutions

  • 1999–2014
    • University of Florida
      • • Department of Chemistry
      • • Department of Biomedical Engineering
      • • Department of Biochemistry and Molecular Biology
      Gainesville, Florida, United States
  • 2008–2013
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
  • 2010
    • Vienna University of Technology
      • Institute of Applied Synthetic Chemistry
      Wien, Vienna, Austria
  • 2005
    • Université du Québec à Montréal
      • Department of Chemistry
      Montréal, Quebec, Canada
  • 1996–1999
    • University of New Brunswick
      • Department of Chemistry
      Fredericton, New Brunswick, Canada