Publications by authors named "Wen-I Luo"

10 Publications

  • Page 1 of 1

Electrochemical Hydroxylation of C-C n-Alkanes by Recombinant Alkane Hydroxylase (AlkB) and Rubredoxin-2 (AlkG) from Pseudomonas putida GPo1.

Sci Rep 2017 08 21;7(1):8369. Epub 2017 Aug 21.

Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.

An unprecedented method for the efficient conversion of C-C linear alkanes to their corresponding primary alcohols mediated by the membrane-bound alkane hydroxylase (AlkB) from Pseudomonas putida GPo1 is demonstrated. The X-ray absorption spectroscopy (XAS) studies support that electrons can be transferred from the reduced AlkG (rubredoxin-2, the redox partner of AlkB) to AlkB in a two-phase manner. Based on this observation, an approach for the electrocatalytic conversion from alkanes to alcohols mediated by AlkB using an AlkG immobilized screen-printed carbon electrode (SPCE) is developed. The framework distortion of AlkB-AlkG adduct on SPCE surface might create promiscuity toward gaseous substrates. Hence, small alkanes including propane and n-butane can be accommodated in the hydrophobic pocket of AlkB for C-H bond activation. The proof of concept herein advances the development of artificial C-H bond activation catalysts.
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http://dx.doi.org/10.1038/s41598-017-08610-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566439PMC
August 2017

Mechanistic Study of the Stereoselective Hydroxylation of [2- H ,3- H ]Butanes Catalyzed by Cytochrome P450 BM3 Variants.

Chemistry 2017 Feb 21;23(11):2571-2582. Epub 2016 Dec 21.

Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.

Engineered bacterial cytochrome P450s are noted for their ability in the oxidation of inert small alkanes. Cytochrome P450 BM3 L188P A328F (BM3 PF) and A74E L188P A328F (BM3 EPF) variants are able to efficiently oxidize n-butane to 2-butanol. Esterification of the 2-butanol derived from this reaction mediated by the aforementioned two mutants gives diastereomeric excesses (de) of -56±1 and -52±1 %, respectively, with the preference for the oxidation occurring at the C-H bond. When tailored (2R,3R)- and (2S,3S)-[2- H ,3- H ]butane probes are employed as substrates for both variants, the obtained de values from (2R,3R)-[2- H ,3- H ]butane are -93 and -92 % for BM3 PF and EPF, respectively; whereas the obtained de values from (2S,3S)-[2- H ,3- H ]butane are 52 and 56 % in the BM3 PF and EPF systems, respectively. The kinetic isotope effects (KIEs) for the oxidation of (2R,3R)-[2- H ,3- H ]butane are 7.3 and 7.8 in BM3 PF and EPF, respectively; whereas KIEs for (2S,3S)-[2- H ,3- H ]butanes are 18 and 25 in BM3 PF and EPF, respectively. The discrepancy in KIEs obtained from the two substrates supports the two-state reactivity (TSR) that is proposed for alkane oxidation in cytochrome P450 systems. Moreover, for the first time, experimental evidence for tunneling in the oxidation mediated by P450 is given through the oxidation of the C-H bond in (2S,3S)-[2- H ,3- H ]butane.
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http://dx.doi.org/10.1002/chem.201603956DOI Listing
February 2017

Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone.

Protein Sci 2014 Jun 17;23(6):833-42. Epub 2014 Apr 17.

Department of Molecular Cardiology, The Cleveland Clinic, Cleveland, Ohio, 44195.

Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.
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http://dx.doi.org/10.1002/pro.2466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093958PMC
June 2014

Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

J Inorg Biochem 2014 May 21;134:118-33. Epub 2014 Feb 21.

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan; Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan. Electronic address:

The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions.
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http://dx.doi.org/10.1016/j.jinorgbio.2014.02.005DOI Listing
May 2014

Identification of the proteins required for fatty acid desaturation in zebrafish (Danio rerio).

Biochem Biophys Res Commun 2013 Nov 5;440(4):671-6. Epub 2013 Oct 5.

Institute of Fisheries Science, National Taiwan University, Taipei 10617, Taiwan; Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan; Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.

Zebrafish Δ-5/Δ-6 fatty acid desaturase (Z-FADS) catalyzes the cascade synthesis of long-chain polyunsaturated fatty acids (PUFAs), thereby playing a pivotal role in several biological processes. In the current study, we report that the Z-FADS protein exists in close proximity to certain cytochrome b5 reductases (CYB5R2 and 3) and elongases (ELOVL2, 4, 5 and 7) on the endoplasmic reticulum, as determined using fluorescence microscopy and fluorescence resonance energy transfer. HeLa cells co-transfected with zebrafish fads and elovl2, 4, and 5 produced docosahexaenoic acid (DHA), as detected by gas chromatography. In addition, immunofluorescence cytochemistry and Western blot data revealed that Z-FADS is present in the mitochondria of HeLa cells. Collectively, our results implicate that Z-FADS, the sole fatty acid desaturase ever been identified in zebrafish, can serve as a universal fatty acid desaturase during lipogenesis.
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http://dx.doi.org/10.1016/j.bbrc.2013.09.127DOI Listing
November 2013

Regioselective hydroxylation of C(12)-C(15) fatty acids with fluorinated substituents by cytochrome P450 BM3.

Chemistry 2013 Oct 11;19(41):13680-91. Epub 2013 Sep 11.

Institute of Chemistry, Academia Sinica, Taipei 115 (Taiwan), Fax: (+886) 2-2783-1237; Department of Chemistry, National Cheng Kung University, Tainan 701 (Taiwan).

We demonstrate herein that wild-type cytochrome P450 BM3 can recognize non-natural substrates, such as fluorinated C12 -C15 chain-length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non-fluorinated substrates, spin-shift measurements suggest that fluoro substituents at the ω-position can facilitate rearrangement of the dynamic structure of the bulk-water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis-Menten constant (Km ), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non-fluorinated counterparts. An enhancement of the turnover frequencies (kcat ) for electron transfer from NADPH to the heme iron and for CH bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme-substrate (ES state) to the corresponding transition state (ES(≠) state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C12 -C15 fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω-position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12-fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high-valent oxoferryl species.
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http://dx.doi.org/10.1002/chem.201302402DOI Listing
October 2013

Kinetic and structural characterization of human mortalin.

Protein Expr Purif 2010 Jul 10;72(1):75-81. Epub 2010 Feb 10.

Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, United States.

Human mortalin is an Hsp70 chaperone that has been implicated in cancer, Alzheimer's and Parkinson's disease, and involvement has been suggested in cellular iron-sulfur cluster biosynthesis. However, study of this important human chaperone has been hampered by a lack of active material sufficient for biochemical characterization. Herein, we report the successful purification and characterization of recombinant human mortalin in Escherichia coli. The recombinant protein was expressed in the form of inclusion bodies and purified by Ni-NTA affinity chromatography. The subsequently refolded protein was confirmed to be active by its ATPase activity, a characteristic blue-shift in the fluorescence emission maximum following the addition of ATP, and its ability to bind to a likely physiological substrate. Single turnover kinetic experiments of mortalin were performed and compared with another Hsp70 chaperone, Thermotogamaritima DnaK; with each exhibiting slow ATP turnover rates. Secondary structures for both chaperones were similar by circular dichroism criteria. This work describes an approach to functional expression of human mortalin that provides sufficient material for detailed structure-function studies of this important Hsp70 chaperone.
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http://dx.doi.org/10.1016/j.pep.2010.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2887293PMC
July 2010

Protein fucosylation regulates synapsin Ia/Ib expression and neuronal morphology in primary hippocampal neurons.

Proc Natl Acad Sci U S A 2006 Jan 22;103(1):21-6. Epub 2005 Dec 22.

Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.

Although fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] carbohydrates have been implicated in cognitive processes such as long-term memory, the molecular mechanisms by which these sugars influence neuronal communication are not well understood. Here, we present molecular insights into the functions of Fucalpha(1-2)Gal sugars, demonstrating that they play a role in the regulation of synaptic proteins and neuronal morphology. We show that synapsins Ia and Ib, synapse-specific proteins involved in neurotransmitter release and synaptogenesis, are the major Fucalpha(1-2)Gal glycoproteins in mature cultured neurons and the adult rat hippocampus. Fucosylation has profound effects on the expression and turnover of synapsin in cells and protects synapsin from degradation by the calcium-activated protease calpain. Our studies suggest that defucosylation of synapsin has critical consequences for neuronal growth and morphology, leading to stunted neurite outgrowth and delayed synapse formation. We also demonstrate that Fucalpha(1-2)Gal carbohydrates are not limited to synapsin but are found on additional glycoproteins involved in modulating neuronal architecture. Together, our studies identify important roles for Fucalpha(1-2)Gal sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity.
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http://dx.doi.org/10.1073/pnas.0503381102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1324972PMC
January 2006

Toxicity assessment of mono-substituted benzenes and phenols using a Pseudomonas initial oxygen uptake assay.

Environ Toxicol Chem 2005 Feb;24(2):253-60

Department of Chemistry, National Cheng Kung University, Tainan, Taiwan 701.

A methodology is presented for assessing the toxicity of chemical substances through their inhibitory action toward the Pseudomonas initial oxygen uptake (PIOU) rate. The current studies reveal that the PIOU assay is rapid, cost-efficient, and easy to perform. The oxygen uptake rate was found to be associated with a putative benzoate transporter and highly dependent on benzoate concentration. The putative benzoate transporter has been shown to follow Michaelis-Menten kinetics. Most phenols were found to be noncompetitive inhibitors of the benzoate transporter. The inhibition constant (Ki) of these noncompetitive inhibitors can be related to the concentration causing 50% oxygen uptake inhibition in Pseudomonas putida. Modeling these data by using the response-surface approach leads to the development of a quantitative structure-activity relationship (QSAR) for the toxicity of phenols ((1/Ki) = -0.435 (+/-0.038) lowest-unoccupied-molecular orbital + 0.517 (+/-0.027)log K(OW) - 2.340 (+/-0.068), n = 49, r2 = 0.930, s = 0.107, r2adj = 0.926, F = 303.1). A comparison of QSAR models derived from the Ki data of the PIOU method and the toxicity data of 40-h Tetrahymena pyrifomis growth inhibition assay (Tetratox) indicated that there was a high correlation between the two approaches (r2 = 0.925).
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http://dx.doi.org/10.1897/04-212r.1DOI Listing
February 2005

The stereospecific hydroxylation of [2,2-2H2]butane and chiral dideuteriobutanes by the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

J Biol Chem 2003 Oct 8;278(42):40658-69. Epub 2003 Aug 8.

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan.

Experiments on cryptically chiral ethanes have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of configuration at the carbon center attacked. This result would seem to rule out a radical mechanism for the hydroxylation chemistry, at least as mediated by this enzyme. The interpretation of subsequent experiments on n-propane, n-butane, and n-pentane has been complicated by hydroxylation at both the pro-R and pro-S secondary C-H bonds, where the hydroxylation takes place. It has been suggested that these results merely reflect presentation of both the pro-R and pro-S C-H bonds to the hot "oxygen atom" species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of configuration. In the present work, we have augmented these earlier studies with experiments on [2,2-2H2]butane and designed d,l form chiral dideuteriobutanes. Essentially equal amounts of (2R)-[3,3-2H2]butan-2-ol and (2R)-[2-2H1]butan-2-ol are produced upon hydroxylation of [2,2-2H2]butane. The chemistry is stereospecific with full retention of configuration at the secondary carbon oxidized. In the case of the various chiral deuterated butanes, the extent of configurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of configuration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet oxene insertion across the C-H bond similar to that previously noted for singlet carbene insertion (Kirmse, W., and Ozkir, I. S. (1992) J. Am. Chem. Soc. 114, 7590-7591). Finally, we discuss how even the oxene insertion mechanism, with "spin crossover" in the transition state, could lead to small amounts of radical rearrangement products, if and when such products are observed. A scheme is described that unifies the two extreme mechanistic limits, namely the concerted oxene insertion and the hydrogen abstraction radical rebound mechanism within the same over-arching framework.
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http://dx.doi.org/10.1074/jbc.M301018200DOI Listing
October 2003