Critical Glutamic Acid Residues Affecting the Mechanism and Nucleotide Specificity of Vibrio Harveyi Aldehyde Dehydrogenase

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 activity and a pre-steady-state burst of NAD(P)H were also eliminated, implicating Glu253 in promoting the nucleophilicity of the cysteine residue(Cys289) involved in forming the thiohemiacetal intermediate in the enzyme mechanism. Mutation of Glu377 to Gln (E377Q mutant) selectively decreased the k,,, for NAD+-dependent ALDH activity (> 10Z-fold) compared to only a 6-fold loss in NADP+-dependent activity without comparable changes to the K,,, values for substrates. Consequently, the E377Q mutant had a very high specificity for NADP+(k,,,/K, > 10' of that for NAD') which was over 20 times higher than that of the wild-type ALDH. Although a pre-steady-state burst of NAD(P)H was eliminated by this mutation, thioesterase activity was completely retained. Using [ 1 -'H]acetaldehyde as a substrate, a significant deuterium isotope effect was observed, implicating Glu377 in the hydride transfer step and not in acylation or release of the acyl group from the cysteine nucleophile. The increase in specificity of the E377Q mutant for NADP' is consistent with a change in the rate-limiting step determining k,,, from nucleotide-dependent NAD(P)H dissociation to hydride transfer. The results provide biochemical evidence that the two highly conserved Glu residues are involved in different functions in the active site of V harveyi ALDH.Keywords: NAD(P)+ : aldehyde dehydrogenase: Vibrio hnweyi ; mutagenesis. Fatty aldehyde dehydrogenase from Mbrio harveyi (Vh.ALDH), a homodimer of I10 kDa, is a NADP+-specific enzyme which catalyzes the oxidation of long-chain aldehydes. The Vh. ALDH gene encodes a protein of 510 amino acids with some characteristics similar to mammalian class 3 ALDHs even though it has low sequence similarity (<25% identity). In particular, Vh. ALDH has a truncated N-terminal and extended Cterminal domain (Vedadi et al., 1995) with a dimeric structure and can utilize both NAD.' as well as NADP ' (Bognar and Meighen, 1978) as can class 3 ALDHs (Lindahl, 1992;Hempel et al., 1993). However, the kLJKm, for Vh. ALDH with NADP' is about 40 times higher than that for NAD' with a low K,, (1.4 pM) for NADP', in contrast to other ALDHs which do not exhibit this unique preference for NADP'. The specificity of the enzyme for long chain aliphatic aldehydes is reflected in a decrease in K , as the chain length of the aldehyde is increased with kLJKrr, being 2.8 X lo4 higher for dodecanal than acetaldehyde and the k,,, values being relatively constant over this rangeCorrespondence fo E.

Structural Basis of Allosteric Regulation and Substrate Specificity of the Non-Phosphorylating Glyceraldehyde 3-Phosphate Dehydrogenase from Thermoproteus tenax

“…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 Å away from the S g of Cys302 supporting the idea that it functions via an activated water molecule.…”

The Crystal Structure of A Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight into the Reaction Mechanism and Shows A Novel Binding Mode of the 2′-Phosphate of NADP+ and A Novel Cation Binding Site

“…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 Å, 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.…”

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

“…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.…”