CONTROL OF METABOLIC FLUX THROUGH THE QUINATE PATHWAY IN ASPERGILLUS-NIDULANS

The quinic acid utilization (gut) pathway in Aspergillus nidulans is a dispensable carbon utilization pathway that catabolizes quinate to pr otocatechuate via dehydroquinate and dehydroshikimate (DHS). At the us ual 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 actio n of the enzymes quinate dehydrogenase, 3-dehydroquinase and DHS dehyd ratase. The extent of control on metabolic flux exerted by the permeas e and the three pathway enzymes was investigated by applying the techn iques 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 inferre d by use of the summation theorem. These measurements implied that, un der the standard growth conditions used, the values for the flux contr ol coefficients of the components of the quinate pathway were: quinate permease, 0.43; quinate dehydrogenase, 0.36; dehydroquinase, 0.18; DH S dehydratase, < 0.03. Attempts to partially decouple quinate permease from the control over flux by measuring flux at pH 3.5 (when a signif icant percentage of the soluble quinate is protonated and able to ente r the mycelium without the aid of a permease) led to an increase of ap prox. 50 % in the flux control coefficient for dehydroquinase. Taken t ogether with the fact that A. nidulans has a very efficient pH homoeos tasis mechanism, these experiments are consistent with the view that q uinate permease exerts a high degree of control over pathway flux unde r the standard laboratory growth conditions at pH 6.5. The enzymes qui nate dehydrogenase and 3-dehydroquinase have previously been overprodu ced in Escherichia coli, and protocols for their purification publishe d. The remaining gut pathway enzyme DHS dehydratase was overproduced i n E. coli and a purification protocol established. The purified DHS de hydratase was shown to have a K-m of 530 mu M for its substrate DHS an d a requirement for bivalent metal cations that could be fulfilled by Mg2+, Mn2+ or Zn2+. All three gut pathway enzymes were purified in bul k 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 biva lent cations as appropriate. Using these empirically determined relati ve values, in conjunction with the connectivity theorem, the relative ratios of the flux control coefficients for the various quinate pathwa y enzymes, and how this control shifts between them, was determined ov er a range of possible metabolite concentrations. These calculations, although clearly subject to caveats about the relationship between kin etic measurements in vitro and the situation in uivo, were able to suc cessfully predict the hierarchy of control observed under the standard laboratory growth conditions. The calculations imply that the hierarc hy of control exerted by the quinate pathway enzymes is stable and rel atively insensitive to changing metabolite concentrations in the range s most likely to correspond to those found in vivo. The effects of sub stituting the type I 3-dehydroquinases from Salmonella typhi and the A . nidulans AROM protein (a pentadomain protein catalysing the conversi on of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate into 5-enolpyruvy lshikimate 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, overproductio n of heterologous dehydroquinase led to a diminution of pathway flux c aused by a lowering of in uivo quinate dehydrogenase levels. With M. t uberculosis, however, quinate dehydrogenase levels increased above tho se of the wild type. We speculate that these changes in quinate pathwa y enzyme activities may be due to changes in the pool sizes of quinate and dehydroquinate.