Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes
This thesis describes structural and binding studies in enzymes from two different organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA). RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologica...
Main Author: | |
---|---|
Format: | Doctoral or Postdoctoral Thesis |
Language: | English |
Published: |
Uppsala universitet, Strukturell molekylärbiologi
2009
|
Subjects: | |
Online Access: | http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-109697 |
id |
ftuppsalauniv:oai:DiVA.org:uu-109697 |
---|---|
record_format |
openpolar |
spelling |
ftuppsalauniv:oai:DiVA.org:uu-109697 2023-05-15T13:52:43+02:00 Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes Ericsson, Daniel 2009 application/pdf http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-109697 eng eng Uppsala universitet, Strukturell molekylärbiologi Uppsala : Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 684 http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-109697 urn:isbn:978-91-554-7638-0 info:eu-repo/semantics/openAccess Mycobacterium tuberculosis ribonucleotide reductase peptide inhibitors fluorescence polarization lipase interfacial activation hydrolase X-ray structure substrate specificity Structural biology Strukturbiologi Doctoral thesis, comprehensive summary info:eu-repo/semantics/doctoralThesis text 2009 ftuppsalauniv 2023-02-23T21:43:56Z This thesis describes structural and binding studies in enzymes from two different organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA). RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation. Doctoral or Postdoctoral Thesis Antarc* Antarctica Uppsala University: Publications (DiVA) |
institution |
Open Polar |
collection |
Uppsala University: Publications (DiVA) |
op_collection_id |
ftuppsalauniv |
language |
English |
topic |
Mycobacterium tuberculosis ribonucleotide reductase peptide inhibitors fluorescence polarization lipase interfacial activation hydrolase X-ray structure substrate specificity Structural biology Strukturbiologi |
spellingShingle |
Mycobacterium tuberculosis ribonucleotide reductase peptide inhibitors fluorescence polarization lipase interfacial activation hydrolase X-ray structure substrate specificity Structural biology Strukturbiologi Ericsson, Daniel Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes |
topic_facet |
Mycobacterium tuberculosis ribonucleotide reductase peptide inhibitors fluorescence polarization lipase interfacial activation hydrolase X-ray structure substrate specificity Structural biology Strukturbiologi |
description |
This thesis describes structural and binding studies in enzymes from two different organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA). RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation. |
format |
Doctoral or Postdoctoral Thesis |
author |
Ericsson, Daniel |
author_facet |
Ericsson, Daniel |
author_sort |
Ericsson, Daniel |
title |
Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes |
title_short |
Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes |
title_full |
Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes |
title_fullStr |
Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes |
title_full_unstemmed |
Tunnels and Grooves : Structure-Function Studies in Two Disparate Enzymes |
title_sort |
tunnels and grooves : structure-function studies in two disparate enzymes |
publisher |
Uppsala universitet, Strukturell molekylärbiologi |
publishDate |
2009 |
url |
http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-109697 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 684 http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-109697 urn:isbn:978-91-554-7638-0 |
op_rights |
info:eu-repo/semantics/openAccess |
_version_ |
1766257192771518464 |