Structural Studies on the Type II Dehydroquinase From Mycobacterium tuberculosis

Gourley, David G (1996) Structural Studies on the Type II Dehydroquinase From Mycobacterium tuberculosis. PhD thesis, University of Glasgow.

Abstract

The enzyme 3-dehydroquinase (3-dehydroquinate dehydratase; E.C. 4.2.1.10) catalyses the dehydration of 3-dehydroquinate to 3-dehydroshikimate. This dehydration reaction is a common step in two important pathways: the biosynthetic shikimate pathway and the catabolic quinate pathway. Dehydroquinases fall into two distinct classes (type I and type II) which are distinguished by non- homologous amino-acid sequences and biophysical criteria. This thesis describes the solution of the three-dimensional structure of the type II dehydroquinase from Mycobacterium tuberculosis by X-ray crystallography. A new purification protocol was designed for M. tuberculosis dehydroquinase to give protein pure enough for crystallisation. Crystals were grown by the sitting drop method in 12% (w/v) PEG 6000 at pH 7.1 using a protein concentration of 20mug/ml. These crystals diffract beyond 2.0 A. Soaking these crystals in Hg(CN)2 and K2Pt(Cl)4 gave derivatives for calculating phases using multiple isomorphous replacement. These phases were then subjected to improvement using density modification techniques. A map was calculated and a model was built into the map. This model was then refined using data from 30 to 2.0 A to give an overall R-factor of 16.6% and an R-free of 21.5%. The type II dehydroquinase from M. tuberculosis is a dodecamer. The enzyme subunit has an (a/P) type fold. It has a central twisted wall of parallel beta-sheet protected on both sides by two helices. The oligomeric structure of the type II dehydroquinase is a dodecamer. The subunits combine to give an arrangement consisting of four trimers packed tetrahedrally. A model for substrate binding to the type II dehydroquinase which is consistent with the biochemical and crystallographic data is presented and experiments to test this model are suggested.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: N W Isaacs
• Keywords: Organic chemistry, Biochemistry
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75358
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 20:26
• Last Modified: 19 Nov 2019 20:26
• URI: https://theses.gla.ac.uk/id/eprint/75358