The mechanism of discrimination between oxidized and reduced coenzyme in the aldehyde dehydrogenase domain of Aldh1l1

Abstract

Aldh1l1, also known as 10-formyltetrahydrofolate dehydrogenase (FDH), contains the carboxy-terminal domain (Ct-FDH), which is a structural and functional homolog of aldehyde dehydrogenases (ALDHs). This domain is capable of catalyzing the NADP+-dependent oxidation of short chain aldehydes to their corresponding acids, and similar to most ALDHs it has two conserved catalytic residues, Cys707 and Glu673. Previously, we demonstrated that in the Ct-FDH mechanism these residues define the conformation of the bound coenzyme and the affinity of its interaction with the protein. Specifically, the replacement of Cys707 with an alanine resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme. We suggested that this was due to the loss of a covalent bond between the cysteine and the C4N atom of nicotinamide ring of NADP+ formed during Ct-FDH catalysis. To obtain further insight into the functional significance of the covalent bond between Cys707 and the coenzyme, and the overall role of the two catalytic residues in the coenzyme binding and positioning, we have now solved crystal structures of Ct-FDH in the complex with thio-NADP+ and the complexes of the C707S mutant with NADP+ and NADPH. This study has allowed us to trap the coenzyme in the contracted conformation, which provided a snapshot of the conformational processing of the coenzyme during the transition from oxidized to reduced form. Overall, the results of this study further support the previously proposed mechanism by which Cys707 helps to differentiate between the oxidized and reduced coenzyme during ALDH catalysis.

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

“…The authors showed that there is a transient covalent bond between the sulfur atom of the catalytic cysteine and the C4N atom of the nicotinamide ring, which holds the coenzyme in the active “hydride transfer” conformation in the structure of the enzyme with NADP+ (PDB entry 2O2Q). This bond was shown to be an indicator of the oxidation state of the coenzyme [10, 23]. The same adduct was predicted by quantum chemical calculations for ALDH2 [24].…”

“…The mechanism, by which the enzyme differentiates the oxidation states of the coenzyme, is not fully understood. This mechanism was studied in detail by Tsybovsky and coworkers for 10-formyltetrahydrofolate dehydrogenase from Rattus norvegicus (C t -FDH) [6, 23]. The authors showed that there is a transient covalent bond between the sulfur atom of the catalytic cysteine and the C4N atom of the nicotinamide ring, which holds the coenzyme in the active “hydride transfer” conformation in the structure of the enzyme with NADP+ (PDB entry 2O2Q).…”

Thiol switches in mitochondria: operation and physiological relevance

“…Aldehyde dehydrogenases, and in particular Aldh2, have been known to contain redox-sensitive thiol groups that are subject to oxidative inactivation (Loomes and Kitson, 1989;Moon et al, 2005;Wang et al, 2011) (Figure 4A). Recent studies on the reaction mechanism of aldehyde dehydrogenases indicated a transient covalent bond of the conserved reactive cysteine residue with the nicotinamide ring of NADP + (DiazSanchez et al, 2011;Tsybovsky et al, 2013).…”

Modeling of interactions between functional domains of ALDH1L1

“…This residue is buried in the core of each C-terminal subunit (~15 Å from the protein surface), and accessible only via two channels that approach the sulfhydryl from opposite sides (14,19,20). One channel holds the electron acceptor, NADP + , while the other presumably provides access to the S-formylated 4-PP electron donor.…”

“…We suggest that two most distinct conformations in this spectrum of conformations are closed (where the ACP domain is associated with the dehydrogenase domain and the 4-PP group inserted into the dehydrogenase catalytic center) and fully open (the formyltransferase, ACP, and dehydrogenase domains are dissociated from each other). While the closed conformation is suggested by the necessity to bring the 4-PP S-formyl moiety in proximity with the dehydrogenase catalytic cysteine (14,19,20,35), the nature of the extended conformation is less obvious. Such an extended conformation, however, is suggested not only by the multicenter ALDH1L1 catalytic mechanisms, but also by the necessity for the ACP domain to interact with 4-PP transferase, the only enzyme in humans that adds the 4-PP to acyl carrier proteins (36,37).…”