Role of residues Asp161, Arg164 and Glu211 in Mycobacterium tuberculosis NADPH-ferredoxin reductase

Mycobacterium tuberculosis NADPH-ferredoxin reductase (NFR or FprA)1, is a FAD-containing oxidoreductase, homologous to mammalian adrenodoxin reductase (AdR). The bacterial enzyme has been proposed to play a major role in the lipid metabolism of the pathogen, as a source of reduced ferredoxin(s) to sustain the activity of its several cytochrome P450s. The resolution of the crystal structure2 of NFR allowed us to identify two ionisable residues (D161 and E211) located in the active site and potentially involved in catalysis of hydride transfer. In order to evaluate the role of these residues, we produced six variants of the enzyme carrying various single point mutations (E211A, E211D, E211Q, D161A, D161E, D161N). All NFR forms were overproduced in E. coli We were able to isolate all E211 mutants in holoenzyme form. Their functional characterization indicated that E211 plays a significant but non-essential role in substrate binding, resulting in a moderate contribution to catalysis. On the contrary, the variants carrying a substitution at position 161 could only be isolated as apoenzymes. Indeed, all D161 variants undergo loss of FAD prosthetic group during purification, making impossible any kinetic characterization. Moreover, attempts to reconstitute the holoenzyme adding exogenous FAD were unsuccessful. These results indicate a critical role for the D161 side chain in the stabilization of the NFR native conformation and in FAD binding. Since the side chain of D161 forms a salt bridge with R164, it was argued that replacement of the former residue with a neutral one, could induce a reorganization of the orphan Arg side-chain, resulting in destabilization of the native conformation. Thus, two additional NFR variants were produced, carrying either a single mutation (R164A) or a double mutation (D161A/R164A). Both mutants turned out to be highly unstable, suggesting that both member of the ionic couple D161/R164 are critical for the stability of the enzyme. These results indicate that most of the ionisable residues located at active site of NFR are more important for maintain the native conformation of the enzyme than for its catalytic activity. 1. Fischer F. et al. (2002) EUR. J. BIOCHEM. 269, 3005-3013. 2. Bossi R.T. et al. (2002) BIOCHEMISTRY 41, 8807-8818.