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Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.

The effect of viral infection on the regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in Nicotiana tabacum L. leaves was studied. PEPC activity was 3 times higher in infected plant leaves compared to healthy plants. Activity of plant PEPC can be regulated, e.g., by de novo synthesis or reversible phosphorylation. The reason for the increase of PEPC activity as a consequence of PVY(NTN) infection was studied. The amount of PEPC determined by Western blot analysis or by relative estimation of PEPC mRNA by real-time PCR did not differ in control and PVY(NTN)-infected plants. Changes in posttranslational modification of PEPC by phosphorylation were evaluated by comparing activity of the native and the dephosphorylated enzyme. The infected plants were characterized by a higher decrease of the enzyme activity after its dephosphorylation, which indicated a higher phosphorylation level. Immunochemical detection of phosphoproteins by Western blot analysis showed a more intensive band corresponding to PEPC from the infected material. This strengthens the hypothesis of an infection-related phosphorylation, which could be part of the plant's response to pathogen attack. The physiological implications of the increase in PEPC activity during PVY(NTN) infection are discussed.

Malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40, NADP-ME), which was found in chloroplasts, was isolated from tobacco leaves (Nicotiana tabacum L.) almost homogenous. The specific enzyme activity was 0.95 μmol min-1 mg-1. The enzyme pH optimum was found between pH 7.1 and 7.4. The affinity of NADP-ME to substrates (L-malate and NADP+) was evaluated in the presence of divalent metal ions (Mg2+, Mn2+, Co2+, Ni2+). The value of the apparent Michaelis constant of NADP-ME for L-malate was dependent on the ion cofactor, while no such relationship was found for NADP+. The dependence of the reaction rate on concentration of Mg2+ indicates the presence of more than one binding site for these ions in NADP-ME. Likewise, the sigmoidal dependence of the reaction rate on Mn2+ concentration and the value of Hill coefficient 7.5 indicate the positive cooperativity of the reaction kinetics in the presence of the ions. The effect of Co2+ and Ni2+ ions was analogous to that of Mn2+ ions; however, the cooperativity was lower (the values of Hill coefficients were 3.0 and 1.3 for Co2+ and Ni2+, respectively). Regulation of NADP-ME from tobacco leaves by divalent metal ions is discussed.