University of St Andrews
In this study, several aspects of nitrate assimilation and transport have been studied using the filamentous fungus Aspergillus nidulans, which has been shown to be safe laboratory organism as judged by it's pathogenicity towards insect larvae. In silico analysis of the A. nidulans genome sequence, identified two putative genes designated cnxL and cnxK that might be involved in molybdenum cofactor (a component of nitrate reductase) biosynthesis as well as two putative nitrate reductases encoding genes niaB and niaC. All four genes are hitherto unknown. Although many features of these proteins provided clues of functionality, biochemical and genetical approaches employed in this present study failed to elicit expression of any of these four genes. A NrtA protein structure model was developed based on residue homology with the E. coli GlpT a protein, the structure of which has been solved. The results of thiol cross-linking of three double cysteine mutants in four NrtA essential residues, R87, R368, N168 and N459, indicated that the molecular distance between R87 and R368 is ~ 0.4 Ã…, R368 and N168 ~ 6.2 Ã…, R87 and N459 is ~ 2.2 Ã…. Another important observation was the change in the confirmation of Tm 2 and Tm 8 in the presence of nitrate. This shift resulted in an increase of ~ 2 Ã… gap between the residues R87 and R368. Distances between amino acid residue pairs estimated using such molecular rulers contradicted the NrtA existing model. Cysteine-scanning mutagenesis studies were extended to the generation of a library of single cysteine mutants of NrtA residues spanning Tm 2 and Tm 8. The majority of single cysteine mutants possessed wild type NrtA protein expression levels but unfortunately most were found to be loss-of-function. Consequently, thiol chemistry of this crop of mutants was not perused. Attempts were also made to overexpress and crystallise the bacterial nitrate transporters but none of the transporter tested proved to be a successful candidate for crystallisation. In this regard, bacterial nitrate transporters, NarU (E. coli), Nar (Bacillus cereus), NarK1 and NarK2 (Pseudomonas aeruginosa) and NarK2 (Thermus thermophilus) fused with GFP were expressed in E. coli and used in crystallisation trials. Although this approach has proved successful for a number of membrane proteins, unfortunately was not helpful with regard to the purification of any of the above bacterial nitrate transporters to yield protein expression levels required for successful protein crystallography. Finally, the effects of potential nitrate transport inhibitors were studied on net nitrate transport by NrtA and NrtB proteins of A. nidulans. The results indicated that chlorate had more of an inhibitory effect on NrtA net nitrate transport than that by NrtB. Chlorite and sulphite equally affected net nitrate transport by either NrtA or NrtB proteins while caesium strongly inhibited the net nitrate transport by NrtB transporter.