7 results on '"Ferguson, Kyle L."'
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2. Informing Efforts to Develop Nitroreductase for Amine Production.
- Author
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Miller, Anne-Frances, Park, Jonathan T., Ferguson, Kyle L., Pitsawong, Warintra, and Bommarius, Andreas S.
- Subjects
NITROREDUCTASES ,AROMATIC amines ,MONOMERS ,BIOCATALYSIS ,FLAVINS - Abstract
Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
3. Evidence for a 1,3-Dipolar Cyclo-addition Mechanism in the Decarboxylation of Phenylacrylic Acids Catalyzed by Ferulic Acid Decarboxylase.
- Author
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Ferguson, Kyle L., Eschweiler, Joseph D., Ruotolo, Brandon T., and Marsh, E. Neil G.
- Subjects
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RING formation (Chemistry) , *DECARBOXYLATION , *FLAVIN mononucleotide , *CINNAMIC acid , *FERULIC acid - Abstract
Ferulic acid decarboxylase catalyzes the decarboxylation of phenylacrylic acid using a newly identified cofactor, prenylated flavin mononucleotide (prFMN). The proposed mechanism involves the formation of a putative pentacyclic intermediate formed by a 1,3 dipolar cyclo-addition of prFMN with the α--β double bond of the substrate, which serves to activate the substrate toward decarboxylation. However, enzymecatalyzed 1,3 dipolar cyclo-additions are unprecedented and other mechanisms are plausible. Here we describe the use of a mechanism-based inhibitor, 2-fluoro-2-nitrovinylbenzene, to trap the putative cyclo-addition intermediate, thereby demonstrating that prFMN can function as a dipole in a 1,3 dipolar cyclo-addition reaction as the initial step in a novel type of enzymatic reaction. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
4. Mechanism of the Novel Prenylated Flavin-Containing Enzyme Ferulic Acid Decarboxylase Probed by Isotope Effects and Linear Free-Energy Relationships.
- Author
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Ferguson, Kyle L., Arunrattanamook, Nattapol, and Marsh, E. Neil G.
- Subjects
- *
ISOPRENYLATION , *ISOTOPIC analysis , *FREE energy (Thermodynamics) , *FLAVINS , *DECARBOXYLASES , *FERULIC acid , *ENZYMATIC analysis - Abstract
Ferulic acid decarboxylase from Saccharomyces cerevisiae catalyzes the decarboxylation of phenylacrylic acid to form styrene using a newly described prenylated flavin mononucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the αβ bond of the substrate that serves to activate the substrate toward decarboxylation. We measured a combination of secondary deuterium kinetic isotope effects (KIEs) at the α- and β-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent KIE is 3.3 on Vmax/KM but is close to unity on Vmax, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the α- and β-positions but vary in magnitude depending on whether the reaction is performed in H2O or D2O. In D2O, the enzyme catalyzed the exchange of deuterium into styrene; this reaction was dependent on the presence of bicarbonate. This observation implies that CO2 release must occur after protonation of the product. Further information was obtained from a linear free-energy analysis of the reaction through the use of a range of para- and meta-substituted phenylacrylic acids. Log(kcat/KM) for the reaction correlated well with the Hammett σ- parameter with ρ = −0.39 ± 0.03; r² = 0.93. The negative ρ value and secondary isotope effects are consistent with the rate-determining step being the formation of styrene from the prenylated flavin-product adduct through a cyclo-elimination reaction. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
5. Steam Explosion (STEX) of Citrus × Poncirus Hybrids with Exceptional Tolerance to Candidatus Liberibacter Asiaticus (CLas) as Useful Sources of Volatiles and Other Commercial Products.
- Author
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Dorado, Christina, Bowman, Kim D., Cameron, Randall G., Manthey, John A., Bai, Jinhe, and Ferguson, Kyle L.
- Subjects
CITRUS greening disease ,CANDIDATUS liberibacter asiaticus ,CITRUS ,ORANGES ,CITRUS fruits ,ALTERNATIVE crops - Abstract
Simple Summary: Orange and grapefruit production in Florida has dropped 75% because of a disease known as Huanglongbing (HLB) which is caused by a bacteria (CLas). Infected trees produce immature, green fruit and eventually die. Methods to treat CLas are very expensive and there is no cure. Many of the valuable products found in oranges and grapefruits can be found in citrus hybrid varieties and these hybrid varieties are known to be naturally very tolerant to CLas. In this work, we used steam treatment followed by water washing for the recovery of valuable citrus products from three citrus hybrids, US-802, US-897, and US-942. For most of the products, more than 80% could be recovered from the hybrids using steam treatment followed by water washing. These citrus hybrids, therefore, have the potential to be an alternative citrus crop that thrives in an HLB environment producing economically valuable products that are recovered in high yields in an environmentally friendly way. Florida citrus production has declined 75% due to Huanglongbing (HLB), a disease caused by the pathogenic bacterium Candidatus Liberibacter asiaticus (CLas). Methods to combat CLas are costly and only partially effective. The cross-compatible species Poncirus trifoliata and some of its hybrids are known to be highly tolerant to CLas, and thus can potentially serve as an alternative feedstock for many citrus products. To further investigate the commercial potential of citrus hybrids, three citrus hybrids, US-802, US-897, and US-942, were studied for their potential as feedstocks for citrus co-products using steam explosion (STEX) followed by water extraction. Up to 93% of sugars were recovered. US-897 and US-942 have similar volatile profiles to that of the commercial citrus fruit types and as much as 85% of these volatiles could be recovered. Approximately 80% of the pectic hydrocolloids present in all three hybrids could be obtained in water washes of STEX material. Of the phenolics identified, the flavanone glycosides, i.e., naringin, neohesperidin, and poncirin were the most abundant quantitatively in these hybrids. The ability to extract a large percentage of these compounds, along with their inherent values, make US-802, US-897, and US-942 potentially viable feedstock sources for citrus co-products in the current HLB-blighted environment. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
6. Analysis and Potential Value of Compounds Extracted From Star Ruby, Rio Red, and Ruby Red Grapefruit, and Grapefruit Juice Processing Residues via Steam Explosion.
- Author
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Dorado C, Cameron RG, Manthey JA, Bai J, and Ferguson KL
- Abstract
Culled whole grapefruit (WG) and grapefruit juice processing residues (GP) are currently incorporated into low-cost animal feed. If individual chemical components found within these side streams could be recovered as high-value coproducts, this would improve the overall value of the grapefruit crop. In this study, pectic hydrocolloids, sugars, volatiles, phenolics, and flavonoids were extracted from Star Ruby, Rio Red, and Ruby Red GP and WG using a continuous pilot scale steam explosion system. Up to 97% of grapefruit juice oils and peel oils could be volatilized and contained 87-94% d -limonene. The recovery of pectin, as determined by galacturonic acid content, was between 2.06 and 2.72 g 100 g
-1 . Of the phenolics and flavonoids analyzed in this study, narirutin and naringin were extracted in the amounts of up to 10,000 and 67,000 μg g-1 , respectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dorado, Cameron, Manthey, Bai and Ferguson.)- Published
- 2021
- Full Text
- View/download PDF
7. Isofunctional enzymes PAD1 and UbiX catalyze formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase.
- Author
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Lin F, Ferguson KL, Boyer DR, Lin XN, and Marsh EN
- Subjects
- Benzoates chemistry, Benzoates metabolism, Carboxy-Lyases chemistry, Carboxy-Lyases genetics, Catalysis, Cinnamates metabolism, Coenzymes chemistry, Escherichia coli genetics, Escherichia coli Proteins chemistry, Flavin Mononucleotide chemistry, Flavin Mononucleotide metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Spectrophotometry, Ultraviolet, Carboxy-Lyases metabolism, Coenzymes metabolism, Escherichia coli Proteins metabolism
- Abstract
The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.
- Published
- 2015
- Full Text
- View/download PDF
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