dc.contributor.advisor | Sarma, Siddhartha P | |
dc.contributor.author | Bansal, Akanksha | |
dc.date.accessioned | 2021-01-04T09:31:38Z | |
dc.date.available | 2021-01-04T09:31:38Z | |
dc.date.submitted | 2018 | |
dc.identifier.uri | https://etd.iisc.ac.in/handle/2005/4789 | |
dc.description.abstract | This thesis ellucidates the mechanism of activation and regulation of AHAS I
enzyme from Escherichia coli that was characterised using crystallographic and
sedimentation velocity analytical ultracentrifugation studies.
AHAS I is a large multi-subunit enzyme that was discovered in the 1950’s, by
Umbarger, as the enzyme that catalyses the first committed step in the biosynthesis
of branched chain amino acids, isoleucine, leucine and valine in bacteria,
plants and certain fungi. The enzyme itself is composed of a catalytic (large)
subunit (CSU / LSU) and a regulatory (small) subunit (RSU / SSU) that are
coded for by the genes IlvB and IlvN, respectively. The enzyme also requires TPP,
FAD and Mg2+ as co-factors. The enzyme catalyses the thiamine dependent decarboxylation
of pyruvate followed by an “acyloin like" condensation with either
another molecule of pyruvate or with 2-ketobutyrate. The product of the former
condensation is acetolactate, while that of the later is 2-keto-2-hydroxybutyrate,
which are precursors for the synthesis of valine (and leucine) and isoleucine,
respectively. Feedback regulation occurs via binding of valine or isoleucine to
the RSU, wherein the amino acids are negative and positive effectors of the enzyme,
respectively. The LSU’s are well conserved across species. The RSU’s on
the other hand differ significantly in sequence and size across species and in the
selectivity towards inhibition by one of the branched chain amino acids. The catalytic
and enzymatic properties of this enzyme have been studied in great detail. Despite this rich history, structures of the AHAS holoenzyme from any species
have been elusive and as a consequence the structural basis for the activation
and regulation of this enzyme remain unclear at best. It has been surmised that
the intrinsic instability of the holoenzyme complex, as a result of weak interactions
between the AHAS subunits, results in heterogeneity of the holoenzyme
preparation and that this is the major factor affecting its crystallisation. On the
other hand structural characterization of the holoenzyme using solution NMR
methods is a daunting task due to the inherent size of the complex. However
the RSU has been successfully cloned, purified and characterized using NMR.
Important information regarding the behaviour of IlvN in the effector bound
and free state has been elucidated. The structural differences as observed for
IlvN in the presence of valine as opposed to isoleucine have also been partially
characterised.
Even with the availability of these data, understanding the difference in affinity
exhibited by IlvN for the two effector molecules remains undescribed due
to the lack of structures elucidating effector binding and its interactions with
residues in the binding pocket. This thesis describes the crystallographic and analytical
ultracentrifugation studies of the subunits of the E.coli AHAS I enzyme
and of the AHAS I holoenzyme. The crystallographic studies were carried out to
understand at atomic resolution the conformational factors that determine selectivity
for the effector molecules. The hydrodynamic studies were carried out to
understand the solution conformational properties and the oligomeric status of
the subunits in solution and the stoichiometry of the subunits in the catalytically
active holoenzyme. | en_US |
dc.language.iso | en_US | en_US |
dc.relation.ispartofseries | G29666 | |
dc.rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part
of this thesis or dissertation | en_US |
dc.subject | holoenzyme | en_US |
dc.subject | E coli | en_US |
dc.subject | hydrodynamic studies | en_US |
dc.subject.classification | Research Subject Categories::NATURAL SCIENCES::Biology::Organism biology::Microbiology | en_US |
dc.title | Crystallographic and analytical ultracentrifugation studies of the E. coli acetohydroxy acid synthase I enzyme | en_US |
dc.type | Thesis | en_US |
dc.degree.name | PhD | en_US |
dc.degree.level | Doctoral | en_US |
dc.degree.grantor | Indian Institute of Science | en_US |
dc.degree.discipline | Faculty of Science | en_US |