Engineering the substrate binding site of benzoylformate decarboxylase.

Abstract:

:Benzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are both thiamin diphosphate-dependent enzymes. The two share a common three-dimensional structure and catalyze a similar chemical reaction, i.e., decarboxylation of 2-keto acids. However, they vary significantly in their substrate utilization pattern. In particular, BFDC has extremely limited activity with pyruvate, while PDC has no activity with benzoylformate. Here we report our progress, using a semirandom approach, toward converting BFDC into an efficient pyruvate decarboxylase. From the structure of BFDC in complex with R-mandelate, 12 residues within a 5 A radius from the inhibitor molecule were selected and subjected individually to site-saturation mutagenesis. Each variant was screened for its ability to decarboxylate five different substrates, i.e., benzoylformate, 2-ketohexanoate, 2-ketopentanoate, 2-ketobutanoate, and pyruvate. The first round of mutagenesis showed that changes in Thr377 and Ala460 resulted in an altered substrate spectrum which included higher activity toward pyruvate. Two variants, T377L and A460Y, were selected as the starting point of a second round of site-saturation mutagenesis. In both cases, the T377L-A460Y double mutant proved to be the only new variant with significantly improved catalytic activity toward pyruvate. When compared to the wild-type enzyme, based on k(cat)/K(m) values, the T377L-A460Y variant showed an 11000-fold improvement in the ratio between pyruvate and benzoylformate utilization. This double mutant displays a K(m) value for pyruvate of 2 mM as well as a k(cat)/K(m) value for pyruvate which is only 70-fold lower than that of Zymomonas mobilis PDC.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Yep A,McLeish MJ

doi

10.1021/bi9008402

subject

Has Abstract

pub_date

2009-09-08 00:00:00

pages

8387-95

issue

35

eissn

0006-2960

issn

1520-4995

journal_volume

48

pub_type

杂志文章
  • Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity.

    abstract::Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid pep...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.5b00392

    authors: Shin A,Lee E,Jeon D,Park YG,Bang JK,Park YS,Shin SY,Kim Y

    更新日期:2015-06-30 00:00:00

  • Chemically Induced Changes to Membrane Permeability in Living Cells Probed with Nonlinear Light Scattering.

    abstract::Second-harmonic light scattering (SHS) permits characterization of membrane-specific molecular transport in living cells. Herein, we demonstrate the use of time-resolved SHS for quantifying chemically induced enhancements in membrane permeability. As proof of concept, we examine the enhanced permeability of the cytopl...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.5b00600

    authors: Wilhelm MJ,Sharifian Gh M,Dai HL

    更新日期:2015-07-28 00:00:00

  • Effect of an Ala81His mutation on the Met80 loop dynamics of iso-1-cytochrome c.

    abstract::An A81H variant of yeast iso-1-cytochrome c is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome c, which has evolved from Ala in yeast to Ile in mammals, slows the dynamics of the opening of the heme crevice. The A81H mutation is used both to increase steric ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi501252z

    authors: Bandi S,Bowler BE

    更新日期:2015-03-10 00:00:00

  • A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein.

    abstract::Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. An extensive analysis of the de...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi991923d

    authors: Konno T,Kamatari YO,Tanaka N,Kamikubo H,Dobson CM,Nagayama K

    更新日期:2000-04-11 00:00:00

  • Exonuclease-polymerase active site partitioning of primer-template DNA strands and equilibrium Mg2+ binding properties of bacteriophage T4 DNA polymerase.

    abstract::The binding of bacteriophage T4 DNA polymerase (T4 pol) to primer-template DNA with 2-aminopurine (2AP) located at the primer terminus results in the formation of a hyperfluorescent 2AP state. Changes in this hyperfluorescent state were utilized to investigate the fractional concentration of primer-templates bound at ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi980074b

    authors: Beechem JM,Otto MR,Bloom LB,Eritja R,Reha-Krantz LJ,Goodman MF

    更新日期:1998-07-14 00:00:00

  • Sequential DNA Binding and Dimerization Processes of the Photosensory Protein EL222.

    abstract::EL222 is a blue light sensor protein, which consists of a light-oxygen-voltage domain as a light sensor and a LuxR-type helix-turn-helix DNA-binding domain. The reaction dynamics of the protein-DNA binding were observed for the first time using the time-resolved transient grating method. The reaction scheme was determ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.7b01206

    authors: Takakado A,Nakasone Y,Terazima M

    更新日期:2018-03-13 00:00:00

  • Restricted motion of the lipoyl-lysine swinging arm in the pyruvate dehydrogenase complex of Escherichia coli.

    abstract::The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase (PDH) complex of Escherichia coli house the lipoyl-lysine side chain essential for active-site coupling and substrate channelling within the complex. The structure of the unlipoylated form of the innermost ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi992978i

    authors: Jones DD,Stott KM,Howard MJ,Perham RN

    更新日期:2000-07-25 00:00:00

  • Prion protein selectively binds copper(II) ions.

    abstract::The infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear. Usi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi972827k

    authors: Stöckel J,Safar J,Wallace AC,Cohen FE,Prusiner SB

    更新日期:1998-05-19 00:00:00

  • Conformational transition required for efficient splicing of transcripts from hybrid lambda promoter yeast tRNA gene fusion.

    abstract::Fusion of a prokaryotic promoter to a yeast tRNA gene provides a means for uncoupling analyses of mutations affecting splicing from requirements for transcription and other processing steps. For this purpose, a phage lambda promoter was fused to the Saccharomyces cerevisiae tRNATyr(SUP3a) coding sequence. This fusion ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00240a017

    authors: Shapero MH,Greer CL

    更新日期:1991-07-02 00:00:00

  • Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST.

    abstract::Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi101776t

    authors: Hwang S,Schmitt AA,Luteran AE,Toone EJ,McCafferty DG

    更新日期:2011-02-01 00:00:00

  • Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide.

    abstract::Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi060176z

    authors: Ohya M,Nishitani C,Sano H,Yamada C,Mitsuzawa H,Shimizu T,Saito T,Smith K,Crouch E,Kuroki Y

    更新日期:2006-07-18 00:00:00

  • Calorimetric studies of adenosine 5'-triphosphate hydrolysis by heavy meromyosin.

    abstract::The heat production for the reaction steps of ATP hydrolysis by heavy meromyosin has been reexamined by using a reaction-type calorimeter. So far, the values reported for the heat production have varied substantially among research groups. The present results obtained in 0.3 M KCl, 10 mM MgCl2, and 20 mM Tris-HCl at p...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00518a037

    authors: Yamada T,Shimizu H,Suga H

    更新日期:1981-07-21 00:00:00

  • Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.

    abstract::Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulf...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9912950

    authors: Eubanks S,Lu M,Peyton D,Breslow E

    更新日期:1999-10-12 00:00:00

  • All-atom molecular dynamics simulations of actin-myosin interactions: a comparative study of cardiac α myosin, β myosin, and fast skeletal muscle myosin.

    abstract::Myosins are a superfamily of actin-binding motor proteins with significant variations in kinetic properties (such as actin binding affinity) between different isoforms. It remains unknown how such kinetic variations arise from the structural and dynamic tuning of the actin-myosin interface at the amino acid residue le...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi4006896

    authors: Li M,Zheng W

    更新日期:2013-11-26 00:00:00

  • Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of N-alkyl and N-alkenyl heme adduct formation.

    abstract::Incubation of 3-(2-phenylethyl)-4-methylsydnone (PMS) with liver microsomes from phenobarbital-pretreated rats or with reconstituted cytochrome P-450b results in loss of the enzyme chromophore. Chromophore loss is NADPH-dependent even though the sydnone decomposes by an oxygen- but not enzyme-dependent process to give...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00413a034

    authors: Grab LA,Swanson BA,Ortiz de Montellano PR

    更新日期:1988-06-28 00:00:00

  • Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase.

    abstract::The crystal structure of an extracellular triglyceride lipase (from a fungus Rhizomucor miehei) inhibited irreversibly by diethyl p-nitrophenyl phosphate (E600) was solved by X-ray crystallographic methods and refined to a resolution of 2.65 A. The crystals are isomorphous with those of n-hexylphosphonate ethyl ester/...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00120a034

    authors: Derewenda U,Brzozowski AM,Lawson DM,Derewenda ZS

    更新日期:1992-02-11 00:00:00

  • Structure of Ralstonia eutropha flavohemoglobin in complex with three antibiotic azole compounds.

    abstract::Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi101650q

    authors: El Hammi E,Warkentin E,Demmer U,Limam F,Marzouki NM,Ermler U,Baciou L

    更新日期:2011-02-22 00:00:00

  • Spectroscopic Investigation of Cysteamine Dioxygenase.

    abstract::Thiol dioxygenases are mononuclear non-heme FeII-dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rat...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.0c00267

    authors: Fernandez RL,Dillon SL,Stipanuk MH,Fox BG,Brunold TC

    更新日期:2020-07-07 00:00:00

  • Peptide models of protein folding initiation sites. 2. The G-H turn region of myoglobin acts as a helix stop signal.

    abstract::A series of peptide fragments of sperm whale myoglobin, corresponding to segments of the region between the G- and H-helices of the protein, have been synthesized and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00076a007

    authors: Shin HC,Merutka G,Waltho JP,Wright PE,Dyson HJ

    更新日期:1993-06-29 00:00:00

  • Systematic evaluation of candidate ligands regulating ectodomain shedding of amyloid precursor protein.

    abstract::Despite intense interest in the proteolysis of the β-Amyloid Precursor Protein (APP) in Alzheimer's disease, how the normal processing of this type I receptor-like glycoprotein is physiologically regulated remains ill-defined. In recent years, several candidate protein ligands for APP, including F-spondin, Reelin, β1 ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi400165f

    authors: Rice HC,Young-Pearse TL,Selkoe DJ

    更新日期:2013-05-14 00:00:00

  • Formation of short-lived protein aggregates directly from the coil in two-state folding.

    abstract::Recent results on the 102 residue protein U1A show that protein aggregation is not always slow and irreversible but may take place transiently in refolding studies on a millisecond time scale. In this study we observe a similar aggregation behavior with the classical two-state protein CI2. Since both U1A and CI2 appea...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9909997

    authors: Silow M,Tan YJ,Fersht AR,Oliveberg M

    更新日期:1999-10-05 00:00:00

  • Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1.

    abstract::Transporters from the SLC13 family couple the transport of two to four Na+ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na+/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue Vc...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.7b00503

    authors: Colas C,Schlessinger A,Pajor AM

    更新日期:2017-08-22 00:00:00

  • NMR constraints on the location of the retinal chromophore in rhodopsin and bathorhodopsin.

    abstract::Rhodopsin is the photoreceptor in vertebrate rod cells responsible for vision at low light intensities. The photoreactive chromophore in rhodopsin is 11-cis-retinal bound to the protein via a protonated Schiff base with Glu113 as the counterion. We have used the observed 13C NMR chemical shifts of the conjugated retin...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00004a037

    authors: Han M,Smith SO

    更新日期:1995-01-31 00:00:00

  • Transfer RNA-pseudouridine synthetase Pus1 of Saccharomyces cerevisiae contains one atom of zinc essential for its native conformation and tRNA recognition.

    abstract::RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant P...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi972671o

    authors: Arluison V,Hountondji C,Robert B,Grosjean H

    更新日期:1998-05-19 00:00:00

  • Mutagenesis of the uncoupling protein of brown adipose tissue. Neutralization Of E190 largely abolishes pH control of nucleotide binding.

    abstract::For expression in Saccharomyces cerevisiae the cDNA of the uncoupling protein (UCP) of brown adipose tissue from hamster has been isolated and used to transform yeast cells. Optimized expression conditions yielded 2% of mitochondrial protein as UCP. UCP was isolated, avoiding copurification of ADP/ATP carrier and pori...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi970513r

    authors: Echtay KS,Bienengraeber M,Klingenberg M

    更新日期:1997-07-08 00:00:00

  • Binding of proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy.

    abstract::A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00458a036

    authors: Kalb E,Engel J,Tamm LK

    更新日期:1990-02-13 00:00:00

  • The three-dimensional structure of the gallium complex of azoverdin, a siderophore of Azomonas macrocytogenes ATCC 12334, determined by NMR using residual dipolar coupling constants.

    abstract::In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi025990a

    authors: Wasielewski E,Atkinson RA,Abdallah MA,Kieffer B

    更新日期:2002-10-15 00:00:00

  • Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase.

    abstract::Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Previous work has demonstrated that the enzyme cycles between a catalytically inactive open state and a catalytically active closed state. The transition of the enzyme between the...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.5b01215

    authors: Johnson TA,Mcleod MJ,Holyoak T

    更新日期:2016-01-26 00:00:00

  • Identification of Cys139 and Glu207 as catalytically important groups in the active site of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

    abstract::Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the antarafacial [1.3] allylic rearrangement of isopentenyl diphosphate (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Active-site thiols at C138 and C139 were recently identified by covalent modification u...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00180a014

    authors: Street IP,Coffman HR,Baker JA,Poulter CD

    更新日期:1994-04-12 00:00:00

  • Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions.

    abstract::Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are com...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00591a032

    authors: Neubig RR,Cohen JB

    更新日期:1979-11-27 00:00:00