Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism

Abstract

Xanthine dehydrogenase, a molybdenum, iron-sulfur tlavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type 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 [L157P]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, LE89Klxanthine 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 proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]xanthine dehydrogenase and IS357FIxanthine dehydrogenase mutated in the flavin domain, and [GI 01 IEIxanthine 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 [G353D]xanthine dehydrogenase, there was an increase in apparent K,, values both for NAD ' and NADH. These findings indicate involvement of this part of the sequence in the NAD+-binding site. The G l O l l 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 centre. However, it retains 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 xanthine and pterin. 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 molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romiio, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270, 1170-11761 from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [GI 01 IEIxanthine 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 activit...

Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme

“…In other batches the specific activity of the recombinant enzyme in the xanthine : PMS\cytochrome c assay was generally lower, e.g. 30 % of the values for the enzyme purified from fruit flies [18].…”

“…Data summarizing the activity of a highly active batch of the recombinant enzyme are compared with published data for the purified endogenous fruit fly enzyme [18] in Table 2. The recombinant enzyme sample has the same activity as the enzyme purified from Drosophila in the xanthine : PMS\cytochrome c assay and marginally less in the xanthine : NAD + assay.…”

“…The UV\visible spectra of samples of recombinant XDH are shown in Figure 1, in comparison with bovine milk XO [6] and wild-type XDH purified [18] from the fruit fly D. melanogaster. Though all the spectra are similar to one another, subtle differences were revealed by deconvolution analysis (see the Materials and methods section).…”

“…For the recombinant enzyme, all three signals are also clearly detectable ( Figure 2B, trace e). However, there [18]. Xanthine : PMS/ cytochrome c (cyt c), xanthine : NAD + and acetaldehyde : NAD + assays were performed in triplicate under standard conditions, as described in the text.…”

“…It does, nevertheless, yield a heterogeneous mixture of a number of different forms of the enzyme, of which apparently no more than 10 % corresponds to the native functional enzyme form [15]. Recombinant expression in Drosophila [13] resulted in only very low levels of expression, estimated to be only 10 % of the nonrecombinant level, making the purification of enzyme on a worthwhile scale (see [18]) from such a system quite impractical.…”