| Abstrakt: |
Xanthine dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaste by the rosy gene has been characterised both from the withtype and mutant flies Enzyme assays. using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [ L127F] and [L1 57P]xanthine dehydrogenases (in all of which the mutation is in the iron -sulfur domain), the enzyme molecules. although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme. [E8QK] xanthine dehydrogenase. These both had ultraviolet -visible absorption spectra similar to milk xanthine oxidase Both were found to be quite stable molecules showing very high catalytic centre activities and with little tendency to become degraded by proteoIysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D] xantine dehydrogenase and [S357F]xanthine dehydrogenase mutated in the flavin domain, and [G1011E] xanthine dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter In addition for [G53D] xanthine dehydrogenase. there was an increase in apparent Km, values both for NAD and NADH. These findings indicate involvement of this part of the sequence in the NAD -binding site. The G1011 E mutation has a profound effect on the enzyme As isolated and as present in crude extracts of the flies, this xanthine dehydrogenase variant lacks activity to xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum center. However, it remain full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it apparently irreversibly to a form showing full activity to xantine and petrin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybedenum cofactor from the tetrahyro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Rom&asim;o M. J., Archer , M., Moura, I., Moura, J.J.G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Hubber, R. (1995) Science 270, 1170-1176] from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio giagas. It is poposed, that apparent irreversibility of the oxidative activating process for [G1011E] xantine dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding ptering incorporation into xanthine dehydrogenase and in relation to other molybdenum enzymes are discussed. [ABSTRACT FROM AUTHOR] |
