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
Biochemistryjournal_title
Biochemistryauthors
Yep A,McLeish MJdoi
10.1021/bi9008402subject
Has Abstractpub_date
2009-09-08 00:00:00pages
8387-95issue
35eissn
0006-2960issn
1520-4995journal_volume
48pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全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
更新日期:2015-06-30 00:00:00
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
更新日期:2015-07-28 00:00:00
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
更新日期:2015-03-10 00:00:00
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
更新日期:2000-04-11 00:00:00
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
更新日期:1998-07-14 00:00:00
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
更新日期:2018-03-13 00:00:00
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
更新日期:2000-07-25 00:00:00
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
更新日期:1998-05-19 00:00:00
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
更新日期:1991-07-02 00:00:00
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
更新日期:2011-02-01 00:00:00
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
更新日期:2006-07-18 00:00:00
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
更新日期:1981-07-21 00:00:00
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
更新日期:1999-10-12 00:00:00
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
更新日期:2013-11-26 00:00:00
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
更新日期:1988-06-28 00:00:00
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
更新日期:1992-02-11 00:00:00
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
更新日期:2011-02-22 00:00:00
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
更新日期:2020-07-07 00:00:00
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
更新日期:1993-06-29 00:00:00
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
更新日期:2013-05-14 00:00:00
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
更新日期:1999-10-05 00:00:00
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
更新日期:2017-08-22 00:00:00
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
更新日期:1995-01-31 00:00:00
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
更新日期:1998-05-19 00:00:00
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
更新日期:1997-07-08 00:00:00
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
更新日期:1990-02-13 00:00:00
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
更新日期:2002-10-15 00:00:00
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
更新日期:2016-01-26 00:00:00
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
更新日期:1994-04-12 00:00:00
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
更新日期:1979-11-27 00:00:00