Kinetics and Thermostability of NADP-Isocitrate Dehydrogenase from Cephalosporium acremonium

Abstract

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 ...

Chemical modification of NADP‐isocitrate dehydrogenase from Cephalosporium acremonium

“…Thus, the limiting inactivation rate constant in the presence of d s ‐isocitrate or the free Mg 2 + ions was about 3.5‐fold lower than that observed in its absence. Furthermore, these results indicated the binding of free d s ‐isocitrate and free Mg 2 + ions to the enzyme independently of each other, as was previously reported from the results of protection effect against thermal inactivation [9]. It was also evident that the presence of Mg 2 + ions reinforced binding of isocitrate ( K s for Mg 2 + d s ‐isocitrate complex is sixfold lower than the K s for d s ‐isocitrate).…”

“…NADP + ‐isocitrate dehydrogenase from the mycelium of C. acremonium CW‐19 ( Acremonium chrysogenum ATCC 36225) grown essentially as described previously [10] was purified according the method of Olano et al . [9]. Fractions containing homogeneous enzyme, as assessed by SDS/PAGE, were pooled and dialyzed for 24 h at 4 °C against 5 m m potassium phosphate buffer (pH 6.5) containing 5 m m potassium citrate, 2 m m MgCl 2 and 40% glycerol, after which it was stored at −20 °C until use.…”

“…Protein concentration was measured by the method of Bradford [23], with bovine serum albumin as a standard. Molar concentrations of NADP‐isocitrate dehydrogenase were estimated for dimers of 140 kDa [9].…”

“…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].…”

Influence of carbon and nitrogen source on synthesis of NADP+-isocitrate dehydrogenase, methylmalonyl-coenzyme A mutase, and methylmalonyl-coenzyme A decarboxylase in Saccharopolyspora erythraea CA340

“…NADP -isocitrate dehydrogenase was assayed as reported by Olano et al [11]. Activity was expressed as Wmols of NADPH formed per mg protein per minute.…”