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Detection of the time-course of thiamin-bound intermediates on enzymatic pathways using steady state and time-resolved spectroscopy

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Title
Detection of the time-course of thiamin-bound intermediates on enzymatic pathways using steady state and time-resolved spectroscopy
Name (type = personal)
NamePart (type = family)
Patel
NamePart (type = given)
Hetalben
NamePart (type = date)
1984-
DisplayForm
Hetalben Patel
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Jordan
NamePart (type = given)
Frank
DisplayForm
Frank Jordan
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Huskey
NamePart (type = given)
Phil
DisplayForm
Phil Huskey
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Pietrangelo
NamePart (type = given)
Agostino
DisplayForm
Agostino Pietrangelo
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Meyers
NamePart (type = given)
Caren Freel
DisplayForm
Caren Freel Meyers
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
outside member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
Graduate School - Newark
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (qualifier = exact)
2014
DateOther (qualifier = exact); (type = degree)
2014-01
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Abstract (type = abstract)
Since the discovery of thiamine diphosphate (ThDP, active form of vitamin B1) as active cofactor in 1937 by Lohmann and Schuster, its catalytic mechanism has become an interest of many enzymologist. The ThDP-dependent enzymes catalyze a variety of enzymatic reactions including decarboxylation, condensation, ligation etc. Here is reported a mechanistic study carried out with multiple ThDP enzymes: Escherichia coli 1-deoxy- D-xylulose-5-phosphate (DXP) synthase, E. coli E1 component (E1o) of the 2-oxoglutarate dehydrogenase complex (OGDHc), yeast pyruvate decarboxylase (YPDC) and benzaldehyde lyase (BAL) by using state-of-the art instruments: the circular dichroism (CD) method can monitor events on the enzyme itself followed by pre-steady state rate determination by stopped-flow (SF) CD, the NMR method monitors ThDP-bound intermediates after release from the enzyme (fortuitously all the major ones are stable in acid), and is also useful to provide positive identification of the species seen in the CD spectrum. Several of these enzymes have in common initial pyruvate decarboxylation to form aldehyde or carboligation products. Steady state and pre-steady state analysis with the enzyme DXP synthase, enabled observation of remarkable stabilization of ThDP-bound pre-decarboxylation intermediate C2α-lactylThDP (LThDP) on the enzyme with pyruvate, and subsequent LThDP decarboxylation greatly accelerated by the second substrate D-glyceraldehyde-3-phosphate (GAP) by at least 600-fold. Further stabilization of LThDP was observed on DXP synthase variants Y392F, R478A and R420A which are shown to be essential for binding of D-GAP, without affecting the LThDP decarboxylation rates in the presence of GAP. While DXP synthase stabilizes pre-decarboxylation intermediate LThDP, the enzyme E1o from E. coli on decarboxlation of 2-oxoglutarate stabilizes the resulting enamine intermediate followed by hydroxy-carboxypropylidene-ThDP radical formation (with both substrates 2-oxoglutarate and 2-oxoadipate), characterized by CD, SFCD, NMR and electron paramagnetic resonance (EPR) spectroscopy. The E. coli E1o was also used in the chiral synthesis of α-hydroxy ketones with good yield and high enantiomeric excess, by varying both the 2-oxoacid substrates and exogenous aldehydes. Next, The conjugated 2-oxoacid substrates acetyl pyruvate, its methyl ester and (E)-4-(4 chlorophenyl)-2-oxo-3-butenoic acid (CPB) with thiamin enzymes YPDC, BAL and E1o led to formation of charge transfer bands on the enzymes which are attributed to transitions between the thiazolium ring of thiamin and a C2-β, γ double bond.
Subject (authority = RUETD)
Topic
Chemistry
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_5164
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
xx, 134 p. : ill.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = vita)
Includes vita
Note (type = statement of responsibility)
by Hetalben Patel
Subject (authority = ETD-LCSH)
Topic
Thiamin pyrophosphate
Subject (authority = ETD-LCSH)
Topic
Thiamin pyrophosphate-dependent enzymes
RelatedItem (type = host)
TitleInfo
Title
Graduate School - Newark Electronic Theses and Dissertations
Identifier (type = local)
rucore10002600001
Location
PhysicalLocation (authority = marcorg); (displayLabel = Rutgers, The State University of New Jersey)
NjNbRU
Identifier (type = doi)
doi:10.7282/T3Z03680
Genre (authority = ExL-Esploro)
ETD doctoral
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Rights

RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Patel
GivenName
Hetalben
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (point = start); (qualifier = exact)
2013-11-08 16:26:01
AssociatedEntity
Name
Hetalben Patel
Role
Copyright holder
Affiliation
Rutgers University. Graduate School - Newark
AssociatedObject
Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
RightsEvent
Type
Embargo
DateTime (encoding = w3cdtf); (point = start); (qualifier = exact)
20140320
DateTime (encoding = w3cdtf); (point = end); (qualifier = exact)
2016-01-31
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after January 31, 2016.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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ContentModel
ETD
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