Control of metabolic flux through the quinate pathway in Aspergillus nidulans
Authors: WHEELER Kerry A. , LAMB Heather K. , HAWKINS Alastair R. 
Published: August 2015

The quinic acid utilization (qut) pathway in Aspergillus nidulans is a dispensable carbon utilization pathway that catabolizes quinate to protocatechuate via dehydroquinate and dehydroshikimate (DHS). At the usual in vitro growth pH of 6.5, quinate enters the mycelium by means of a specific permease and is converted into PCA by the sequential action of the enzymes quinate dehydrogenase, 3-dehydroquinase and DHS dehydratase. The extent of control on metabolic flux exerted by the permease and the three pathway enzymes was investigated by applying the techniques of Metabolic Control Analysis. The flux control coefficients for each of the three quinate pathway enzymes were determined empirically, and the flux control coefficient of the quinate permease was inferred by use of the summation theorem. These measurements implied that, under the standard growth conditions used, the values for the flux control coefficients of the components of the quinate pathway were: quinate permease, 0.43; quinate dehydrogenase, 0.36; dehydroquinase, 0.18; DHS dehydratase, &lt; 0.03. Attempts to partially decouple quinate permease from the control over flux by measuring flux at pH 3.5 (when a significant percentage of the soluble quinate is protonated and able to enter the mycelium without the aid of a permease) led to an increase of approx. 50% in the flux control coefficient for dehydroquinase. Taken together with the fact that A. nidulans has a very efficient pH homoeostasis mechanism, these experiments are consistent with the view that quinate permease exerts a high degree of control over pathway flux under the standard laboratory growth conditions at pH 6.5. The enzymes quinate dehydrogenase and 3-dehydroquinase have previously been overproduced in Escherichia coli, and protocols for their purification published. The remaining qut pathway enzyme DHS dehydratase was overproduced in E. coli and a purification protocol established. The purified DHS dehydratase was shown to have a Km of 530 μM for its substrate DHS and a requirement for bivalent metal cations that could be fulfilled by Mg2+, Mn2+ or Zn2+. All three qut pathway enzymes were purified in bulk and their elasticity coefficients with respect to the three quinate pathway intermediates were derived over a range of concentrations in a core tricine/NaOH buffer, augmented with necessary cofactors and bivalent cations as appropriate. Using these empirically determined relative values, in conjunction with the connectivity theorem, the relative ratios of the flux control coefficients for the various quinate pathway enzymes, and how this control shifts between them, was determined over a range of possible metabolite concentrations. These calculations, although clearly subject to caveats about the relationship between kinetic measurements in vitro and the situation in vivo, were able to successfully predict the hierarchy of control observed under the standard laboratory growth conditions. The calculations imply that the hierarchy of control exerted by the quinate pathway enzymes is stable and relatively insensitive to changing metabolite concentrations in the ranges most likely to correspond to those found in vivo. The effects of substituting the type I 3-dehydroquinases from Salmonella typhi and the A. nidulans AROM protein (a pentadomain protein catalysing the conversion of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate into 5-enolpyruvylshikimate 3-phosphate), and the Mycobacterium tuberculosis type II 3-dehydroquinase, in the quinate pathway were investigated and found to have an effect. In the case of S. typhi and A. nidulans, overproduction of heterologous dehydroquinase led to a diminution of pathway flux caused by a lowering of in vivo quinate dehydrogenase levels. With M. tuberculosis, however, quinate dehydrogenase levels increased above those of the wild type. We speculate that these changes in quinate pathway enzyme activities may be due to changes in the pool sizes of quinate and dehydroquinate.