Ingrid Kuo

The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold.

The first structure of an aldehyde dehydrogenase (ALDH) is described at 2.6 Å resolution. Each subunit of the dimeric enzyme contains an NAD-binding domain, a catalytic domain and a bridging domain. At the interface of these domains is a 15 Å long funnel-shaped passage with a 6 × 12 Å opening leading to a putative catalytic pocket. A new mode of NAD binding, which differs substantially from the classic β-α-β binding mode associated with the ‘Rossmann fold’, is observed which we term the β-α,β mode. Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family. A mechanism for enzymatic specificity and activity is postulated.

Purification and characterization of a troponin C-like phosphodiesterase activator from bovine thyroid.

A troponin C-like phosphodiesterase activator from bovine thyroid has been purified to homogeneity. The overall purification was about 9,800-fold with a yield of 8%. Bovine thyroid activator protein is identical in biologic properties to that isolated from bovine brain. They have the same specific activity regarding stimulation of bovine brain cyclic nucleotide phosphodiesterase. Both proteins form a Ca2+-dependent complex with heart muscle troponin I which is stable in 6M urea-polyacrylamide gel and which is similar, but not identical, to the troponin C-troponin I complex. The physiochemical properties of bovine thyroid activator protein are identical with those of bovine brain and other phosphodiesterase activator proteins and are similar to heart muscle and skeletal muscle troponin C as follows: (A) they bind 3-4 exchangeable calcium ions/mol with dissociation constants between 10(-5) and 10(-6) M, (B) they are highly acidic with a high content of aspartic and glutamic acids and isoelectric points of approximately 4.1, (C) these proteins have an unusual ultraviolet absorption spectrum with six discrete maxima between 250 and 284 nm which are characteristic of phenylalanine and tyrosine, and (D) these proteins have a low content of cysteine, histidine, tyrosine and proline. The tryptic peptide maps of bovine thyroid and brain activator protein are very similar. However, despite a very similar amino acid composition, the peptide map of bovine heart muscle troponin C is significantly different from that of the other two proteins. The molecular weight of thyroid and brain activator protein is 16,500, while that of heart troponin C is 18,500. Thyroid and brain activator protein, as well as heart troponin C, appear to undergo significant Ca2+-dependent conformational changes, as measured by the difference in the circular dichroism spectrum and electrophoretic mobility observed in the presence and absence of calcium ion.

UDP-glucose dehydrogenase: Substrate binding stoichiometry and affinity

Abstract The direct binding of UDP-glucose and NAD+ to bovine liver UDP-glucose dehydrogenase has been measured by equilibrium dialysis and differential fluorescence. At saturation the hexameric enzyme binds only three molecules each of UDP-glucose and NAD+. The binding of NAD+ is virtually characteristic of that for noninteracting identical sites with a binding constant of about 0.47 × 104. UDP-Glucose, however, binds more avidly than NAD+ and exhibits negative cooperativity characterized by unrestricted Adair constants of 16.1, 3.7, and 0.37, all × 104.

Binding ratios by fluorescence with self-compensation for optical inner filter effects

Abstract A simple procedure which self-compensates for optical inner filter effects has been developed for measuring the binding of ligands to macro-molecules by fluorescence measurements in moderately optically dense solutions. Correction for light source fluctuations during the course of measurement is also automatically achieved, provided the fluctuations are not too frequent, i.e., not greater than one every minute. The method is applicable both to systems involving fluorescing ligans which undergo a quantum efficiency change upon binding and to systems in which the macromolecule contains chromophores whose quantum efficiencies change upon ligand association. Examples of the use of the method with each type of system are presented in the text. The necessary fluorescence parameters are measurable with standard widely available commercial instrumentation. The general approach applied here to single-site proteins can, in principle, be extended to multisite systems as well.

Crystal Structure of a Class 3 Aldehyde Dehydrogenase at 2.6Å Resolution

Class 3 ALDHs prefer NAD as coenzyme, but can use NADP effectively in vitro. They function as dimers of identical ~50kDa monomers, and share about 30% sequence identity with either of the tetrameric class 1 or 2 enzymes. Interest in the Class 3 ALDH (ALDH-3) has focused on its diversity of expression (Lindahl, 1992). No ALDH-3 activity is detectable in normal mammalian liver, but high levels are obtained after exposure to certain xenobiotics. Many neoplasms possess elevated ALDH-3 activities, while many normal tissues, such as cornea and stomach constitutively express ALDH-3. ALDH-3 enzymes prefer aromatic aldehydes and medium chain-length (C-6 to C-10) aliphatic aldehydes as substrate. Known substrates include benzaldehyde and hexenal as well as 4-hydroxynonenal derived from membrane lipid peroxidation (Lindahl and Peterson, 1991). The microsomal ALDH-3 has also received clinical attention from the recent demonstration that Sjogren-Larsson Syndrome is the result of mutations inactivating this “fatty aldehyde dehydrogenase” (DeLaurenzi, et al, 1996).

Beyond the catalytic core of ALDH: a web of important residues begins to emerge

Site-directed mutagenesis was performed in class 3 aldehyde dehydrogenase (ALDH) on both strictly conserved, non-glycine residues, Glu-333 and Phe-335. Both lie in Motif 8 and are indicated to be of central catalytic importance from their positions in the tertiary structure. In addition, a highly conserved residue at the end of Motif 8, Pro-337, and Asp-247, which interacts with the main chain of Motif 8, were also mutated. All substitutions were conservative. Kinetic values clearly show that Glu-333 and Phe-335 are crucial to efficient catalysis, along with Asp-247. Pro-337 appears to have a different role, most likely relating to folding.

Rat Class 3 Aldehyde Dehydrogenase: Crystals and Preliminary Analysis

Class 3 aldehyde dehydrogenase (A1DH) from rat liver has been expressed in native form by E. coli from the pTALDH vector (Harper et al., 1988). This A1DH differs substantially from the class 1 and 2 AlDHs both in catalytic properties and in primary and quaternary structure. However, secondary structural predictions suggest that the subunit tertiary structures of all three A1DH classes are largely similar (Lindahl and Evces, 1984; Hempel et al., 1989). Although functional residues have been identified from chemical modification and sequence comparisons, no tertiary structure of an A1DH has yet been determined.