Uda, Narasimha Rao. Promiscuous behaviour of the bacterial metallohydrolase DapE : an evolutionary and mechanistic perspective. 2015, PhD Thesis, University of Basel, Faculty of Science.
Available under License CC BY-NC-ND (Attribution-NonCommercial-NoDerivatives).
Official URL: http://edoc.unibas.ch/diss/DissB_11346
Here, we investigated the bacterial binuclear metallohydrolase, N-Succinyl-L-LDiaminopimelic acid desuccinylase DapE (EC 188.8.131.52) of S.enterica. DapE is an essential enzyme in the late stage of the lysine biosynthetic pathway that also provides a crucial building block of the peptidoglycan cell wall. Since DapE is essential for most Gram-negative and many Gram-positive bacteria and it is not present in humans, it has been proposed as a very good target for antibiotic development. It was also reported that DapE has a metal dependent promiscuous aspartyl dipeptidase activity, in which incorporation of either zinc or manganese to the enzyme leads to activity with different substrates and this phenomenon occurs both in vivo and in vitro.
We addressed the reaction mechanism of the native desuccinylase activity as well as the Mn2+-dependent aspartyl dipeptidase promiscuous activity of DapE, by investigating a series of substrate analogues and potential inhibitors. We postulated a plausible mechanism for metal-dependent promiscuity, based on subtle differences in coordination preferences between Mn2+ and Zn2+,which may be widely applicable to other enzymes. We revealed why a promising inhibitor of the enzyme in vitro, L-captopril, fails to exert antibiotic activity and
propose broad practical implications of this discovery for drug-design, as well as fundamental evolutionary implications. We described kinetic cooperativity in DapE, which offers clues on structural rearrangements that occur during catalysis and is also of relevance to inhibitor design.
Finally, we explored the evolutionary aspects of functional robustness of native activity over the promiscuous activity using DapE as a model enzyme and addressed the molecular mechanisms underlying the emergence of functional robustness through laboratory evolution.
|Committee Members:||Schwede, Torsten|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Computational & Systems Biology > Bioinformatics (Schwede)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||139 S.|
|Last Modified:||30 Jun 2016 10:58|
|Deposited On:||04 Dec 2015 14:47|
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