Hydride transfer between pyridine nucleotide and FAD in Mycobacterium tuberculosis FprA: role of the invariant His57 in catalysis as studied by protein engineering, stopped-flow kinetics and X-ray crystallography

FprA is a Mycobacterium tuberculosis NADPH-ferredoxin reductase, homologous to mammalian adrenodoxin reductase (AdR). As the eukaryotic counterpart, FprA is presumably involved in supplying electrons, through ferredoxins, to one or more of the pathogen’s cytochromes P450. The two enzymes share many functional (1) and structural (2) properties. The very high resolution (1.05 Å) of its crystal structure (2) makes FprA an ideal target of protein engineering studies. Furthermore, investigations on the FprA enzymology are expected to increase our knowledge of important details of the mycobacterial metabolism. On the basis of crystallographic data (2), it was previously proposed that the highly conserved His57 of FprA is involved in catalysis. The residue was changed to either Gln or Ala through site-directed mutagenesis. Steady-state kinetics showed that substitutions of His57 markedly impaired the catalytic efficiency of FprA for ferredoxin in the physiological reaction. The analysis of the reductive half-reaction of wild-type and His57Gln forms with NADPH and NADH by diode-array stopped-flow spectrophotometry allowed a detailed description of the mechanism of the reduction of the enzyme-bound FAD, with the identification of the charge-transfer intermediates involved. The three-dimensional structure of FprA-H57Q, obtained at 1.8 Å resolution, reveals a sub-optimal geometry of the nicotinamide-isoalloxazine interaction in the active site. These data demonstrate that the side chain of His57 has a role in optimizing the geometry of the enzyme-bound substrate for the subsequent steps of the catalytic cycle to proceed at a high rate. This conclusion is expected to be valid also for mammalian AdR. 1. Fischer, F., Raimondi, D., Aliverti, A., and Zanetti, G. (2002) Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase, Eur. J. Biochem. 269, 3005-3013. 2. Bossi, R. T., Aliverti, A., Raimondi, D., Fischer, F., Zanetti, G., Ferrari, D., Tahallah, N., Maier, C. S., Heck, A. J. R., Rizzi, M., and Mattevi, A. (2002) A covalent modification of NADP+ revealed by the atomic resolution structure of FprA, a Mycobacterium tuberculosis oxidoreductase, Biochemistry 41, 8807–8818.