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Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. (Alwyn Jones)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2009. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 684
Keywords [en]
Mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, fluorescence polarization, lipase, interfacial activation, hydrolase, X-ray structure, substrate specificity
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-109697ISBN: 978-91-554-7638-0 (print)OAI: oai:DiVA.org:uu-109697DiVA, id: diva2:273555
Public defence
2009-12-05, B42, Uppsala Biomedical Center (BMC), Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2009-11-12 Created: 2009-10-22 Last updated: 2012-09-18Bibliographically approved
List of papers
1. Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
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2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 13, no 12, p. 822-832Article in journal (Refereed) Published
Abstract [en]

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that TrP5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain., as compared to Trp5.

Keywords
mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, alanine scan, statistical molecular design, structure activity relationships, FHDoE
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-14261 (URN)10.1002/psc.906 (DOI)000252000600007 ()17918768 (PubMedID)
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2018-01-12Bibliographically approved
2. Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
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2010 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 16, no 3, p. 159-164Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown 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. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors.

Keywords
Fluorescence polarization, Mycobacterium tuberculosis, Peptide inhibitors, Ribonucleotide reductase
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-112344 (URN)10.1002/psc.1214 (DOI)000275448300007 ()20127854 (PubMedID)
Available from: 2010-01-26 Created: 2010-01-13 Last updated: 2018-01-12Bibliographically approved
3. X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
Open this publication in new window or tab >>X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 1, p. 109-119Article in journal (Refereed) Published
Abstract [en]

In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-A resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic alpha/beta hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements 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.

Keywords
lipase, interfacial activation, hydrolase, X-ray structure, substrate specificity
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-14221 (URN)10.1016/j.jmb.2007.10.079 (DOI)000253181500011 ()18155238 (PubMedID)
Available from: 2008-01-29 Created: 2008-01-29 Last updated: 2017-12-11Bibliographically approved

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