Novel NADPH–cysteine covalent adduct found in the active site of an aldehyde dehydrogenase

Abstract

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the 'resting' conformation to the double bond of the α,β-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H-Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

Structural Basis for a Cofactor-dependent Oxidation Protection and Catalysis of Cyanobacterial Succinic Semialdehyde Dehydrogenase

“…example. Such an adduct was predicted by quantum mechanical calculations (27) and then characterized in two different structures from the ALDH family (17,23,28). However, to date, the functional role of the covalent adduct has remained elusive.…”

NADP-Dependent Aldehyde Dehydrogenase from ArchaeonPyrobaculum sp.1860: Structural and Functional Features

“…To the best of our knowledge, the inhibition of the binding of the reactive oxidized form NAD(P) by NAD(P)H was observed only for betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) [21, 22]. The mechanism, by which the enzyme differentiates the oxidation states of the coenzyme, is not fully understood.…”

Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus

“…For the ordered Bi Bi reaction mechanism, substrate inhibition can be caused by the binding of excess substrate to the enzyme-NAD(P) ϩ complex (13,45). Therefore, we performed a UV scanning analysis (280 to 400 nm) of BetB to detect a thio-adduct between its cysteine (Cys289 in BetB) and the pyridine ring of NAD ϩ , which has a typical absorbance peak at 325 nm (8). How- ever, the addition of NAD ϩ to BetB did not result in the increase in absorbance around 325 nm (data not shown), which is consistent with the absence of BetB inhibition by NAD ϩ and suggests that the BetB inhibition is not caused by the formation of a thio-adduct.…”

“…In contrast, the P. aeruginosa BADH (PaBADH, ALDH9) follows a random Bi Bi mechanism, although the mechanism is predominantly ordered in the case of the NADP ϩ -dependent reaction (35). A kinetic study revealed that the activity of PaBADH is inhibited by the cofactor and partially by the aldehyde (25,35), and the structure of this enzyme in complex with the cofactor suggested that this inhibition is caused by a novel NADPH-cysteine covalent adduct (8).…”

“…For dehydrogenases, several molecular mechanisms of substrate inhibition have been proposed, including the formation of a covalent adduct between the oxidized forms of substrate and cofactor, allosteric inhibition (which occurs away from the active site, e.g., in D-3-phosphoglycerol dehydrogenase from Mycobacterium tuberculosis), and the formation of a nonproductive enzyme complex with cofactor and/or substrate (6)(7)(8)(9)(10)(11)(12)(13). The latter mechanism can be associated with the dehydrogenase residues located both in the substrate and in cofactor binding sites.…”