NADP-isocitrate dehydrogenase [isocitrate:NADP ؉ oxidoreductase (decarboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is apparent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg 2؉ is the true substrate. The V max depends on a basic form of an ionizable group of the enzyme-substrate complex with a pK es (pK of the enzyme-substrate complex) of 6.9 and a ⌬H ion (activation enthalpy) of ؊2 ؎ 0.4 kcal mol ؊1 (ca. 8 ؎ 2 kJ mol ؊1). The enzyme showed maximum activity at 60؇C, an unusually high temperature for a nonthermophilic fungus. The thermodynamic parameters for isocitrate oxidative decarboxylation and for the binding of isocitrate and NADP ؉ were calculated. We analyzed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40؇C following a first-order kinetics. The presence of 12 mM Mg 2؉ plus 10 mM DL-isocitrate led to 100% protection of enzyme activity against inactivation at 60؇C for 120 min. Removal of either or both compounds led to activity loss. A greater stabilizing role for Mg 2؉ was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH. Cephalosporium acremonium produces the ␤-lactam antibiotic cephalosporin C. Its biosynthesis from L-␣-aminoadipic acid, L-cysteine, and L-valine has been well characterized (1, 42). Moreover, the gene which codes for the bifunctional protein deacetoxycephalosporin C synthetase-deacetyl-cephalosporin C synthetase was cloned (11, 44). Knowledge of the regulation of the biosynthetic pathway is, however, still incomplete, especially with regard to the control of the precursor side chain of ␣-aminoadipic acid, the starting amino acid for formation of penicillins and cephalosporin C (25). In Cephalosporium spp., the ␣-aminoadipic acid is synthesized from both 2-oxoglutarate and acetyl coenzyme A by a reaction catalyzed by the homocitrate synthetase enzyme. On the other hand, both the deacetoxycephalosporin C synthetase and the deacetoxycephalosporin C hydrolase require 2-oxoglutarate, ascorbate, ferrous ions, and oxygen for good activity (3, 24). The absolute requirement for 2-oxoglutarate in reactions catalyzed by these enzymes shows that its availability in sufficient amounts may be a limiting factor for ring expansion (38). In fungi such as Neurospora crassa and Saccharomyces cerevisiae (37), the formation of 2-oxoglutarate is catalyzed by two enzymes, NAD-isocitrate dehydrogenase (EC 1.1.1.41) and NADP-isocitrate dehydrogenase (EC 1.1.1.42). The NAD-linked enzyme is related mainly to the production of 2-oxoglutarate as an intermediary compound of the Krebs cycle, whereas the NADP-linked enzyme may play an important role in a variety of different biosynthetic metabolic processes. Not only is 2-oxoglutarate a key metabolite, but NADPH formed in a reaction catalyzed by it may contribute ...

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m 21´s21 at pH 6.5 and 25 8C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5,5 H -dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5 H -phosphate reversibly inactivated the enzyme at pH 7.7 and 5 8C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5 H -phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.Keywords: NADP-isocitrate dehydrogenase; chemical modification; Cephalosporium acremonium.Although the NADP + -dependent isocitrate dehydrogenases from pig heart, yeast and Escherichia coli have been extensively studied (reviewed in [1,2]), the complete metabolic functions and several structural features remain to be established. Cephalosporium acremonium produces the b-lactam antibiotic cephalosporin C. The condensation of l-cysteine with l-aaminoadipic acid is the first step in the biosynthetic pathway of all b-lactam antibiotics, where 2-oxoglutarate is not only a precursor of l-a-aminoadipic acid [3], but it is also required in two other steps, those catalyzed by the deacetoxycephalosporin C synthetase and by the deacetoxycephalosporin C hydrolase [4±6]. In C. acremonium, the availability of 2-oxoglutarate has been proposed [7,8] as a limiting factor for cephalosporin C biosynthesis. Due to the potential role of the isocitrate dehydrogenase [threo-d s -isocitrate NADP + oxidoreductase (decarboxylating)] from C. acremonium in supplying 2-oxoglutarate for cephalosporin C biosynthesis, we decided to investigate some regulatory properties of this enzyme. We have shown that it is a dimer of identical subunits, is not allosterically regulated and is unusually stable to temperature in the presence of its true substrate, the magnesium-isocitrate complex [9]. Furthermore, we found maximum values of total NADP-isocitrate dehydrogenase activity in the 4±5 day range on fermentation medium [9], at the time of maximum value of deacetoxycephalosporin C synthetase [10].Knowledge of the role of individual a...

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