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) Biochemistry41, 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.

Purification and kinetic characterization of Haemophilus parasuis malate dehydrogenase.

Haemophilus parasuis malate dehydrogenase ((S)-malate:NAD+ oxidoreductase; EC 1.1.1.37) isolated from cell sonicates was purified 584-fold to electrophoretic homogeneity with a 19% recovery and a specific activity of 222 units/mg protein. SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the purified enzyme to be a dimer composed of 34,600 molecular weight subunits. Kinetic parameters for all four substrates in the forward and reverse reactions indicated a sequential mechanism for this enzymic process. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme and NADH the second product released. Protection against thermodenaturation of the enzyme by NAD and not by malate was supportive of this mechanism. A pronounced product inhibition by NADH (K(i) = 9.0 microM) was observed. Although NADP did not serve as a coenzyme, a number of analogs of NAD structurally altered in the nitrogen base moieties were observed to function as coenzymes in the oxidation of malate catalyzed by the purified malate dehydrogenase. Coenzyme-competitive inhibition of the malate dehydrogenase was observed with five adenosine derivatives and six structural analogs of NAD. Of the NAD analogs studied as inhibitors, 3-pyridylcarbinol adenine dinucleotide was the most effective (K(i) = 18 microM). Although inhibition of growth of H. parasuis by this analog was observed, it was less effective (K(i) = 136 microM) than the inhibition of the purified dehydrogenase.

Hydrophobic chromatography and bioanalysis of some polar pyridine derivatives used as antilipolytic agents.

The antilipolytic compound 5-fluoro-3-pyridinecarboxylic acid and 5-fluoro-3-hydroxymethylpyridine have been determined quantitatively in plasma (Srel = 5-7% in the concentration range 1-20 microgram/ml) by liquid chromatography on LiChrosorb RP-8 (5 micrometer) with phosphate buffer pH 3-4 as mobile phase, after precipitation of proteins and direct injection of the supernatant. Detection limits were 0.1-0.2 microgram/ml. The chromatographic retention is explained by adsorption of the uncharged compounds on to the support complemented by ion-pair adsorption with buffer components at extreme pH values.