No abstract

The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was puri®ed by sizeexclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3±9.6 and 50°C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl¯u oride and chymostatin, but not by soybean trypsin or Bowman±Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin-like. Puri®cation of the enzyme was complicated by the fact that, when puri®ed, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.

No abstract

Fungal infections of barley and wheat cause devastating losses of these food crops. The endogenous proteinase inhibitors produced by plant seeds probably defend the plants from pathogens by inhibiting the degradation of their proteins by the pathogen proteases. We have studied the interactions of barley grain inhibitors with the subtilisin-like and trypsinlike proteinases of Fusarium culmorum. The inhibition kinetics of three inhibitor proteins, chymotrypsin/subtilisin inhibitor 2 (CI-2), barley alpha-amylase/subtilisin inhibitor (BASI), and Bowman-Birk trypsin inhibitor (BBBI), have been studied in detail for the first time using fungal enzymes. The kinetic studies were performed at physiological pH values to mimic in vivo conditions. Numerical approaches to kinetic analyses were used to calculate the inhibition constants, because the data analyses were complicated by some inhibitor turnover and the instability of enzymes and substrates. All were slow, tight-binding inhibitors that followed either a two-step mechanism (CI-2 and BASI) or a single-step mechanism (BBBI) under the conditions investigated. The overall Ki values derived were approximately 50 pM, 1 nM, and 0.1 nM for CI-2, BASI, and BBBI, respectively. The main difference between the CI-2 and the BASI inhibitions was accounted for by the stabilities of their final complexes and the rate constants for their second dissociation steps (9 x 10(-6)/s and 3 x 10(-4)/s, respectively). Understanding the inhibition mechanisms will be valuable in designing improved strategies for increasing the resistance of the grains to fungal infections.