Final Activity Report Summary - OXMEDIAT (Molecular Enzymology of Eicosanoid synthesising enzymes:
Eicosanoids and eicosanoid synthesising enzymes have been implicated in the pathogenesis of various diseases (atherosclerosis, inflammation, osteoporosis, cancer), which are of major health political relevance for all industrialised countries. In most cases these disorders are chronic, gradually crippling and often fatal. The costs, that society has to pay for treatment of these disorders and for the losses in working time, are enormous. In fact, ischemic cardiovascular disorders, which frequently lead to heart attack and stroke, are the major causes of premature lethality in Europe. Hence, these illness cause immense human suffering, shorten life-span and pose an ever increasing burden on the European healthcare system.
The present project was aimed at increasing our knowledge about the structural biology of eicosanoid synthesising enzymes, which are potential targets for drug therapy of these diseases. To achieve our goals we applied an integrated strategy, which involves elements of synthetic organic chemistry, molecular enzymology (recombinant expression and targeted modification of eicosanoid synthesising enzymes by site-directed mutagenesis) and computer-assisted modelling (structural and kinetic modelling and simulation of molecular dynamics). Special attention has been paid to conformational changes of protein matrix (inter domain movement of 12/15-lipoxygenase) in solution and molecular dynamics of oxygen as second lipoxygenase substrate.
Our major finding suggests that mutual interaction of the two domains appears to be crucial for protein stability and its biological function. N-terminal beta-barrel domain of LOXs may serve as built-in modulator of the enzymatic activity and membrane binding. Regarding oxygen movement we obtained strong evidence that the main route for oxygen access to the active site of the enzyme follows a transiently interconnected cavities whereby the opening and closure is governed by side chain dynamics. This path is not conserved and appears to be individual for each particular protein. These data prompt the conclusion that similar structures might exist for many other oxygen-consuming enzymes. This methodology can also be applied to other small hydrophobic molecules.
The present project was aimed at increasing our knowledge about the structural biology of eicosanoid synthesising enzymes, which are potential targets for drug therapy of these diseases. To achieve our goals we applied an integrated strategy, which involves elements of synthetic organic chemistry, molecular enzymology (recombinant expression and targeted modification of eicosanoid synthesising enzymes by site-directed mutagenesis) and computer-assisted modelling (structural and kinetic modelling and simulation of molecular dynamics). Special attention has been paid to conformational changes of protein matrix (inter domain movement of 12/15-lipoxygenase) in solution and molecular dynamics of oxygen as second lipoxygenase substrate.
Our major finding suggests that mutual interaction of the two domains appears to be crucial for protein stability and its biological function. N-terminal beta-barrel domain of LOXs may serve as built-in modulator of the enzymatic activity and membrane binding. Regarding oxygen movement we obtained strong evidence that the main route for oxygen access to the active site of the enzyme follows a transiently interconnected cavities whereby the opening and closure is governed by side chain dynamics. This path is not conserved and appears to be individual for each particular protein. These data prompt the conclusion that similar structures might exist for many other oxygen-consuming enzymes. This methodology can also be applied to other small hydrophobic molecules.