Abstract
The catalytic mechanism of the NAD(P)+-dependent aldehyde dehydrogenases (ALDHs) involves the nucleophilic attack of the essential cysteine (Cys302, mature HsALDH2 numbering) on the aldehyde substrate. Although oxidation of Cys302 will inactivate these enzymes, it is not yet well understood how this oxidation is prevented. In this work we explore possible mechanisms of protection by systematically analyzing the reported three-dimensional structures and amino acid sequences of the enzymes of the ALDH superfamily. Specifically, we considered the Cys302 conformational space, the structure and residues conservation of the catalytic loop where Cys302 is located, the observed oxidation states of Cys302, the ability of physiological reductants to revert its oxidation, and the presence of vicinal Cys in the catalytic loop. Our analyses suggested that: 1) In the apo-enzyme, the thiol group of Cys302 is quite resistant to oxidation by ambient O2 or mild oxidative conditions, because the protein environment promotes its high pKa. 2) NAD(P)+ bound in the “hydride transfer” conformation afforded total protection against Cys302 oxidation by an unknown mechanism. 3) If formed, the Cys302-sulfenic acid is protected against irreversible oxidation. 4) Of the physiological reductant agents, the dithiol lipoic acid could reduce a sulfenic or a disulfide bond in the ALDHs active site; glutathione cannot because its thiol group cannot reach Cys302, and other physiological monothiols may be ineffective in those ALDHs where their active site cannot sterically accommodate two molecules of the monothiols. 5) Formation of the disulfides Cys301-Cys302, Cys302-Cys304, Cys302-Cys305 and Cys-302-Cys306 in those ALDHs that have these Cys residues is not probable, because of the permitted Cys conformers as well as the conserved structure and low flexibility of the catalytic loop. 6) Only in some ALDH2, ALDH9, ALDH16 and ALDH23 enzymes, Cys303, alone or in conjunction with Cys301, allows disulfide formation. Interestingly, several of these enzymes are mitochondrial.
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