Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase

Kitson, Trevor M.; Hill, J. P.; Midwinter, Graeme G.

doi:10.1042/bj2750207

Cited by 42 publications

(27 citation statements)

References 26 publications

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“…For example, we have extensively studied the kinetics of the reaction between cytosolic aldehyde dehydrogenase and certain p-nitrophenyl esters, carbonates, and lactones (1-3), and we have used p-nitrophenyl dimethylcarbamate to provide a label for identifying the enzyme's catalytically essential residue (4). We have developed a very useful cyclic carbamate that provides a covalently linked p-nitrophenol ''reporter group'' and used it to probe the nature of the active site in chymotrypsin (5) and aldehyde dehydrogenase (6).…”

Section: Introductionmentioning

confidence: 99%

Comparison of Resorufin Acetate andp-Nitrophenyl Acetate as Substrates for Chymotrypsin

Kitson

1996

Bioorganic Chemistry

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Section: Introductionmentioning

confidence: 99%

Comparison of Resorufin Acetate andp-Nitrophenyl Acetate as Substrates for Chymotrypsin

Kitson

1996

Bioorganic Chemistry

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“…Further, aldehyde dehydrogenases display half-of-the-sites reactivity with iodoacetamide (Pietruszko et al, 1993), while UDPGDH displays half-of-the-sites reactivity with iodoacetamide and iodoacetic acid (Franzen et al, 1976). In mammalian liver aldehyde dehydrogenases, Cys-302 reacts with iodoacetamide (Hempel et al, 1985) and disulfiram (Kitson et al, 1991) and is the only consensus residue among a large number of distantly related aldehyde dehydrogenase sequences (Hempel et al, 1993).…”

Section: Relationship To Alcohol and Aldehyde Dehydrogenasesmentioning

confidence: 99%

UDP‐glucose dehydrogenase from bovine liver: Primary structure and relationship to other dehydrogenases

et al. 1994

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The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD+-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microheterogeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of -26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases. Because the enzyme combines functionalities of alcohol and aldehyde dehydrogenases, it was also of interest to search specifically for other sequence similarities to either of these 2 enzymes, as well as to histidinol dehydrogenase, another 4-electron-transferring dehydrogenase, but none were found.

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“…ALDH is responsible for both aldehyde dehydrogenase and thioesterase activity (Byers and Meighen, 1984). Cys302 of sheep liver cytoplasmic aldehyde dehydrogenase has been also identified as a catalytically essential nucleophilic residue in esterase activity (Kitson et al, 1991) and simultaneous loss of dehydrogenase and esterase activities on mutation of Glu268 of human liver mitochondria1 and Cys302 of rat liver mitochondria1 ALDH (Farres et al, 1995) has previously been used as support for the involvement of both residues in the formation of the thiohemiacetal intermediate.…”

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confidence: 99%

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

Vedadi

Meighen

1997

European Journal of Biochemistry

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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.

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Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase

Cited by 42 publications

References 26 publications

Comparison of Resorufin Acetate andp-Nitrophenyl Acetate as Substrates for Chymotrypsin

Comparison of Resorufin Acetate andp-Nitrophenyl Acetate as Substrates for Chymotrypsin

UDP‐glucose dehydrogenase from bovine liver: Primary structure and relationship to other dehydrogenases

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

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