Enzymatic Oxidation of NADP+ to its 4-Oxo-Derivative is a Side-Reaction Displayed Only by the Adrenodoxin Reductase Type of Ferredoxin-NADP+ Reductases.

We have previously shown that Mycobacterium tuberculosis FprA, an
NADPH-ferredoxin reductase homologous to mammalian adrenodoxin
reductase, promotes the oxidation of NADP+ to its 4-oxo derivative 3-carboxamide-
4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti
A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS,
Heck AJ, Rizzi M et al. (2002) Biochemistry 41, 8807–8818]. Here, we provide
a detailed study of this unusual enzyme reaction, showing that it
occurs at a very slow rate (0.14 h)1), requires the participation of the
enzyme-bound FAD, and is regiospecific in affecting only the C4 of the
NADP nicotinamide ring. By protein engineering, we excluded the involvement
in catalysis of residues Glu214 and His57, previously suggested to be
implicated on the basis of their localization in the three-dimensional structure
of the enzyme. Our results substantiate a catalytic mechanism for
3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in
which the initial and rate-determining step is the nucleophilic attack of the
nicotinamide moiety by an active site water molecule. Whereas plant-type
ferredoxin reductases were unable to oxidize NADP+, the mammalian
adrenodoxin reductase also catalyzed this unusual reaction. Thus, the
3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction
seems to be a peculiar feature of the mitochondrial type of ferredoxin
reductases, possibly reflecting conserved properties of their active sites.
Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide
phosphate is good ligand and a competitive inhibitor of various dehydrogenases,
making this nucleotide analog a useful tool for the
characterization of the cosubstrate-binding site of NADPH-dependent
enzymes.