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Inhibition, crystal structures, and in-solution oligomeric structure of aldehyde dehydrogenase 9A1.

ABSTRACT: Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD⁺-dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition. Two crystal structures of ALDH9A1 are reported, including the first of the enzyme complexed with NAD⁺. One of the structures reveals the active conformation of the enzyme, in which the Rossmann dinucleotide-binding domain is fully ordered and the inter-domain linker adopts the canonical β-hairpin observed in other ALDH structures. The oligomeric structure of ALDH9A1 was investigated using analytical ultracentrifugation, small-angle X-ray scattering, and negative stain electron microscopy. These data show that ALDH9A1 forms the classic ALDH superfamily dimer-of-dimers tetramer in solution. Our results suggest that the presence of an aldehyde substrate and NAD⁺ promotes isomerization of the enzyme into the active conformation.

SUBMITTER: Wyatt JW

PROVIDER: S-EPMC7484307 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid.

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Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease.

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Inhibition of aldehyde dehydrogenase expands hematopoietic stem cells with radioprotective capacity.

Project description:Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units, verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes.

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Targeting abatacept-resistant T-helper-17 cells by aldehyde dehydrogenase inhibition.

Project description:IL-17-producing helper T (Th17) cells are long-lived and serve as central effector cells in chronic autoimmune diseases. The underlying mechanisms of Th17 persistence remain unclear. We demonstrated that abatacept, a CD28 antagonist, effectively prevented the development of skin disease in a Th17-dependent experimental autoimmune dermatitis model. Abatacept selectively inhibited the emergence of IL-7R-negative effector-phenotype T cells while allowing the survival and proliferation of IL-7R+ memory-phenotype cells. The surviving IL-7R+ Th17 cells expressed genes associated with alcohol/aldehyde detoxification and showed potential to transdifferentiate into IL-7R-negative effector cells. Inhibiting aldehyde dehydrogenase reduced IL-7R+ Th17 cells in vivo, independently of CD28, and exhibited additive effects when combined with abatacept. Our findings suggest that CD28 blockade prevents inflammation without eliminating persistent memory cells. These remaining memory cells can be targeted by other drugs, such as aldehyde dehydrogenase inhibitors, to limit their survival, thereby facilitating the treatment of chronic autoimmune diseases.

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Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant.

Project description:Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.

| S-EPMC1262676 | biostudies-literature

The crystal structure of Aquifex aeolicus prephenate dehydrogenase reveals the mode of tyrosine inhibition.

Project description:TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.

| S-EPMC2676054 | biostudies-literature

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