Studies on bacterial type II dehydroquinases and shikimate dehydrogenases

Evans, Lewis David Brett (2003) Studies on bacterial type II dehydroquinases and shikimate dehydrogenases. PhD thesis, University of Glasgow.

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The enzymes dehydroquinase and shikimate dehydrogenase catalyse the third and fourth steps of the biosynthetic shikimate pathway, respectively. The work in this thesis investigates the active sites and overall structure of bacterial type II dehydroquinases, shikimate dehydrogenases and related proteins using biochemical and biophysical techniques. The active site of Streptomyces coelicolor type II dehydroquinase (SCDHQase) was characterised by site-directed mutagenesis of the residues: Arg113, His106, Glu104, Seri 08 and Tyr28, proposed to be important in previous structural studies of the enzyme. None of the mutations significantly altered the secondary or tertiary structure of the enzyme as shown by circular dichroism. All five mutantions significantly decreased the catalytic activity; the most effective mutation was the Y28F mutation with a 4700-fold reduction in activity. This has provided strong evidence of the role of each of the residues within the active site. To date Bacillus subtilis is the only organism known whose genome encodes proteins that resemble both types of dehydroquinase. The type I dehydroquinase was shown to be the active form in cell extracts of B. subtilis by sodium borohydride trapping of the Schiff base involved in the mechanism of all type I dehydroquinases. The YqhS protein from B. subtilis has 47% identity to the S. coelicolor type II dehydroquinase. However, YqhS has been found to have an extremely low level of dehydroquinase activity (kcat < 10-4 s-1). YqhS has a phenylalanine in place of the highly conserved tyrosine residue found to be catalytically important and implicated in proton abstraction at C2 of the substrate. Site-directed mutagenesis of the phenylalanine to a tyrosine (F23Y) effected a 2000-fold increase in Growth experiments on the disruption mutant of the yqhS gene (YqhSd) produced by the Japanese Bacillus Consortium show that the gene is essential for growth in minimal medium. Detailed studies of the pH dependence, effects of salts on catalytic activity and the inhibitory effect of polyanions were performed on the type II dehydroquinases of S. coelicolor, Mycobacterium tuberculosis (MTDHQase), Helicobacter pylori (HPDHQase) and B. subtilis (F23Y mutant). pH-dependence studies on all the dehydroquinases tested show that there is a seven-fold increase in kcat between pH 6.5 and 8.0 indicating that all of these type II DHQases share the same catalytic mechanism. Low concentrations of chloride cause a moderate increase the catalytic activity of MTDHQase and a 4-fold increase in kcat for HPDHQase. In contrast, the kcat of both SCDHQase and B. subtilis (F23Y mutant) decreases in the presence of low concentrations of chloride. The differences in the effect of chloride are thought to arise from variations in the flexibility of dehydroquinase structures. Polyanions (sulphate and phosphate) are competitive inhibitors of SCDHQase and HPDHQase. The interactions are more complex in MTDHQase and B. subtilis YqhS (F23Y). Analysis of the X-ray structures of SCDHQase and MTDHQase reveals contrasting modes of binding of polyanions between the two enzymes. The differences between the interactions and binding of polyanions in type II dehydroquinases may help to explain the relative potencies of rationally designed inhibitors. The Haemophilus influenzae paralogs; shikimate dehydrogenase (HIAroE) and shikimate dehydrogenase related protein (HIYdiB) have been investigated using steady-state kinetics and detailed analysis of their sequences in relation to the published X-ray structures of Escherichia coli shikimate dehydrogenase and YdiB protein. Both HIAroE and HIYdiB are NADPH-dependent dehydrogenases and catalyse the oxidation of shikimate to form dehydroshikimate, with kcat values of 194 and 19 s-1, respectively. HIAroE is also able to oxidise quinate as part of the catabolic quinate pathway (kcat of 50 s-1). Fluorescence quenching studies have demonstrated that shikimate is able to bind to HIAroE in the absence of cofactor. HIAroE is likely to follow a random sequential mechanism. The function of HIYdiB is still unclear as the catalytic activity of the HIYdiB protein is significantly lower than the HIAroE enzyme for all the substrates tested. The N-terminal amino acid sequence of the HIYdiB protein is significantly different from the E.coli YdiB and other members of the shikimate dehydrogenase family and it is proposed that the N-terminal region defines the function of this protein.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Prof. John Coggins.
Keywords: Microbiology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71422
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 07 Jun 2021 09:13

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