The Potential Roles of the Conserved Amino Acids in Human Liver Mitochondrial Aldehyde Dehydrogenase

Abstract

The sequence alignment of all known aldehyde dehydrogenases showed that only 23 residues were completely conserved (Hempel, J., Nicholas, H., and Lindahl, R. (1993 Oxidation of toxic aldehydes to their corresponding acids is primarily catalyzed by aldehyde dehydrogenase (ALDH).1 During the last two decades, several ALDHs from different organisms were discovered. By aligning the sequences of 16 known ALDHs, it was found that only 23 amino acids were completely conserved (1). More recently, Vasiliou et al. (2) classified 26 mammalian ALDHs based on divergent evolution and reported that same residues were conserved. We undertook a mutagenesis approach to investigate the role of the conserved residues in the human mitochondrial ALDH, which possessed a functional side chain. Since completion of the study, the threedimensional structure of the corresponding beef liver enzyme has been determined to 2.65 Å (3).Prior to the advent of molecular biological techniques, chemical modifications were used to identify the components of the active site of the enzyme. Classical sulfhydryl reagents inactivated the enzyme (4 -6). Iodoacetamide was shown to modify Cys 302 in human ALDH (4) while using protection studies; our laboratory reported that Cys 49 was a component of the active site of the horse liver enzyme (7). Two other residues were identified by chemical modifications as being possibly involved in the catalytic process. These were Glu 268 and Ser 74 in the human (8) and sheep liver (9) enzymes, respectively. It was shown later by site-directed mutagenesis studies that Cys 302 is a nucleophile (10) and Glu 268 (11) functions as a general base during catalysis. However, Ser 74 (12) and Cys 49 /Cys 162 (10) were found to be not essential for the catalytic reaction.The kinetics of ALDH was found to follow an ordered sequential mechanism where NAD ϩ binds first followed by aldehyde (13). The reaction involves both acylation and deacylation steps during the oxidation of aldehyde to acid, or hydrolysis of nitrophenyl acetate. It was proposed that deacylation (k 7 ) (Fig. 1) was rate-limiting for horse liver ALDH2 (13, 14) for the dehydrogenase reaction, while acylation (k 3 ) was rate-limiting for the esterase reaction.Here we report the properties of human ALDH2 mutant enzymes produced by replacing the conserved amino acids possessing a reactive side chain with different residues. The purpose of this study is to understand the potential role of the conserved residues in the catalytic mechanism of ALDH and to relate the effects to the now known structure. In the accompanying paper, a detailed analysis of the Lys 192 and Glu 399 mutants that caused a change in the rate-limiting step of the enzyme will be presented (15). EXPERIMENTAL PROCEDURES Materials-NADϩ and NADH were purchased from Sigma; Sequenase version 2.0 kit was obtained from United States Biochemical Corp.; propionaldehyde, chloroacetaldehyde, and p-nitrophenyl acetate were from Aldrich; Magic Minipreps DNA purification system and T4 DNA ligase were from Promega Corp...

A Disorder to Order Transition Accompanies Catalysis in Retinaldehyde Dehydrogenase Type II

“…Hurley et al (15) have found that mutation of Arg-475 in ALDH2 leads to disorder in the co-factor binding region through a "domino effect." And Sheikh et al (22) observe that mutation of Ser-471 to an alanine results in a 50-fold increase in the K m for NAD. An even more striking effect of a distal mutation on the NAD binding site was described for the naturally occurring ALDH2:E487K mutation.…”

Conserved Catalytic Residues of the ALDH1L1 Aldehyde Dehydrogenase Domain Control Binding and Discharging of the Coenzyme

“…In the case of class 1 and 2 ALDHs, the likely candidate to abstract a proton from Cys-302 is Glu-268 (5,10,20,22). The glutamate then shuttles the proton to bulk water before participating in the deacylation step of the reaction (10,25).…”

“…In the second step, an activated water molecule hydrolyzes the thioester, releasing the product. It has been suggested that deprotonation of the catalytic cysteine prior to the nucleophilic attack on the substrate, a required step in this mechanism, involves acceptance of a proton by either glutamate 268 or 399 (5,7,20,(22)(23)(24). The resulting thiolate ion is likely stabilized by the positively charged nic-otinamide ring of the coenzyme and/or adjacent main chain amide groups.…”

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis

“…The deduced polypeptide consists of 501 amino acids with a predicted molecular mass of 54.3 kD. Its sequence contains the conserved residues that have been identified in Homo sapiens (human) liver ALDH as active site amino acids (Sheikh et al, 1997;Yoshida et al, 1998). The lack of N-or C-terminal extensions as compared with the mammalian liver ALDH suggests that REF1 is likely to be tetrameric (Rodriguez-Zavala and Weiner, 2002).…”