Abstract:Fatty aldehyde dehydrogenase (ALDH) from the luminescent marine bacterium, Vibrio harveyi, differs from other ALDHs in its unique specificity and high affinity for NADP'. Two glutamic acid residues, Glu253 and Clu377, which are highly conserved in ALDHs, were investigated in the present study. Mutation of Glu253 to Ala decreased the k,,, for ALDH activity by over four orders of magnitude without a significant change in the K,, values for substrates or the ability to interact with nucleotides. Both thioesterase… Show more
“…34,35 In accordance with this, a study on ALDH from Vibrio harveyi (similar to mammalian class 3 ALDH) showed that Glu268 increases the nucleophilicity of Cys302 and is thus important in acylation. 36 In contrast, biochemical studies on Sm-GAPN have shown that Glu268 has no impact on the acylation but only on the deacylation step. 2 The carboxyl group of this residue is in several crystal structures of ALDHs located 6 -7 Ă away from the S g of Cys302 supporting the idea that it functions via an activated water molecule.…”
Section: Substrate Binding and Catalysismentioning
“…34,35 In accordance with this, a study on ALDH from Vibrio harveyi (similar to mammalian class 3 ALDH) showed that Glu268 increases the nucleophilicity of Cys302 and is thus important in acylation. 36 In contrast, biochemical studies on Sm-GAPN have shown that Glu268 has no impact on the acylation but only on the deacylation step. 2 The carboxyl group of this residue is in several crystal structures of ALDHs located 6 -7 Ă away from the S g of Cys302 supporting the idea that it functions via an activated water molecule.…”
Section: Substrate Binding and Catalysismentioning
“…9,10,40,42 In the holo-PaBADH crystal, the carboxylate oxygens of E252(E268) and E387(E399) are at distances of 8 and 5 Ă , respectively, away from the sulfur of the catalytic cysteines. E387(E399) has the same conformation present in most of the ALDH structures so far determined; it is hydrogen bonded to the carbonyl oxygen of the main chain of C286(C302) through a water molecule.…”
Section: Structure Determination and Main Characteristicsmentioning
“…The acylation step involves the formation of a hemithioacetal intermediate via the nucleophilic attack of the catalytic Cys-302 (the amino acid numbering used for the biochemical and structural data is that defined by Wang and Weiner (5)) on the aldehydic function followed by hydride transfer that leads to formation of a thioacylenzyme intermediate and NAD(P)H. This intermediate then undergoes a nucleophilic attack by an activated water or CoA molecule. Over the past 15 years both mechanistic and structural aspects of hydrolytic ALDHs have been studied extensively (5)(6)(7)(8)(9)(10)(11)(12)(13). In addition to local conformational reorganizations of the active site induced by ligand binding that provide the required flexibility for an efficient catalysis (14,15), one of the key aspects of the chemical mechanism of this ALDH family is the substantial conformational flexibility of the NMN moiety of the cofactor and in particular of the nicotinamide ring.…”
Section: Structural Dynamics Associated With Cofactor Binding Have Bementioning
Background: Conformational dynamics of the cofactor are essential for catalysis by hydrolytic ALDHs. Results: Crystallographic and kinetic data reveal the molecular basis for NADH release in MSDH, a CoA-dependent ALDH. Conclusion: Weaker stabilization of the adenine ring triggers early NADH release in MSDH-catalyzed reaction. Significance: First description of the mechanism whereby the cofactor binding mode is partly responsible for the kinetic behavior of CoA-dependent ALDHs.
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