10.1021/bi060369m.s001
Andrea Pennati
Andrea
Pennati
Adelia Razeto
Adelia
Razeto
Matteo de Rosa
Matteo de
Rosa
Vittorio Pandini
Vittorio
Pandini
Maria Antonietta Vanoni
Maria Antonietta
Vanoni
Andrea Mattevi
Andrea
Mattevi
Alessandro Coda
Alessandro
Coda
Alessandro Aliverti
Alessandro
Aliverti
Giuliana Zanetti
Giuliana
Zanetti
Role of the His57−Glu214 Ionic Couple Located in the Active Site of
<i>Mycobacterium tuberculosis</i> FprA<sup>†</sup><sup>,</sup><sup>‡</sup>
American Chemical Society
2006
NADH
NADPH
Glu 214
1.8 Å resolution
cytochrome P 450s
FAD
3 D structure
hydride transfer
FprA
2006-07-25 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Role_of_the_His57_Glu214_Ionic_Couple_Located_in_the_Active_Site_of_i_Mycobacterium_tuberculosis_i_FprA_sup_sup_sup_sup_sup_sup_/3068869
<i>Mycobacterium tuberculosis</i> FprA is a NADPH-ferredoxin reductase, functionally and
structurally similar to the mammalian adrenodoxin reductase. It is presumably involved in supplying
electrons to one or more of the pathogen's cytochrome P450s through reduced ferredoxins. It has been
proposed on the basis of crystallographic data (Bossi, R. T., et al. (2002) <i>Biochemistry 41</i>, 8807−8818)
that the highly conserved His57 and Glu214 whose side chains are H-bonded are involved in catalysis.
Both residues were individually changed to nonionizable amino acyl residues through site-directed
mutagenesis. Steady-state kinetics showed that the role of Glu214 in catalysis is negligible. On the contrary,
the substitutions of His57 markedly impaired the catalytic efficiency of FprA for ferredoxin in the
physiological reaction. Furthemore, they decreased the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value for NADPH in the ferricyanide
reduction. Rapid-reaction (stopped-flow) kinetic analysis of the isolated reductive half-reaction of wild-type and His57Gln forms of FprA with NADPH and NADH allowed a detailed description of the
mechanism of enzyme-bound FAD reduction, with the identification of the intermediates involved. The
His57Gln mutation caused a 6-fold decrease in the rate of hydride transfer from either NADPH or NADH
to the enzyme-bound FAD cofactor. The 3D structure of FprA−H57Q, obtained at 1.8 Å resolution,
explains the inefficient hydride transfer of the mutant in terms of a suboptimal geometry of the
nicotinamide−isoalloxazine interaction in the active site. These data demonstrate the role of His57 in the
correct binding of NADPH to FprA for the subsequent steps of the catalytic cycle to proceed at a high
rate.