A Pitcher-and-Catcher Mechanism Drives Endogenous Substrate Isomerization by a Dehydrogenase in Kynurenine Metabolism

Abstract

Edited by Ruma BanerjeeAldehyde dehydrogenase typically performs oxidation of aldehydes to their corresponding carboxylic acid while reducing NAD(P)؉ to NAD(P)H via covalent catalysis mediated by an active-site cysteine residue. These data establish a hidden intrinsic isomerization activity of the dehydrogenase and allow us to propose a pitcher-catcher type of catalytic mechanism for the isomerization.The kynurenine pathway is the catabolic route for tryptophan degradation in mammals and certain bacteria. In mammals, the pathway has been found to produce neuroactive compounds that correlate with depression and neurodegenerative disease states such as Alzheimer's, Parkinson's, and Huntington's diseases (1-3). Moreover, the kynurenine pathway is a de novo biosynthetic route to produce the coenzyme NAD ϩ / NADH, which is involved in many fundamental biological processes as an energy carrier and redox mediator. In the kynurenine pathway, tryptophan metabolites are partitioned by both enzymatic and non-enzymatic reactions (4). Three consecutive enzymes of the pathway, 3-hydroxyanthranilate dioxygenase (HAO), 3 2-amino-3-carboxymuconate-6-semi-aldehyde decarboxylase (ACMSD), and 2-amino-muconate-6-semialdehyde dehydrogenase (AMSDH), compete with the non-enzymatic auto-cyclization of their substrates and products for further metabolism (Fig. 1). The trio of enzymes is also present in the 2-nitrobenzoic acid biodegradation pathway.Investigations at the molecular level of the kynurenine pathway were extended to AMSDH in our recent work (5). AMSDH is a 216-kDa homotetrameric protein (500 amino acid residues in each subunit) that belongs to the aldehyde dehydrogenase superfamily. It competes with a spontaneous, non-enzymatic cyclization of 2-aminomuconate semialdehyde (2-AMS) to prevent overproduction of picolinic acid. The off-pathway product, picolinic acid, is a metal chelator in human milk that is barely detectable in blood serum and below the detection limit in other tissues (6). AMSDH oxidizes 2-AMS to 2-aminomuconate and directs the metabolic flux to enzyme-controlled reactions. We have shown the anticipated enzymatic activity of AMSDH using isolated protein and determined its first crystal structure (5). Furthermore, the binary and ternary complexes as well as two catalytic intermediates, thiohemiacetal and thioacyl, were characterized by soaking single crystals of the binary enzyme-NAD ϩ complex under varied time periods with substrates, widening our knowledge of the catalytic mechanism of this semialdehyde dehydrogenase.

Reassignment of the human aldehyde dehydrogenase ALDH8A1 (ALDH12) to the kynurenine pathway in tryptophan catabolism

“…0.03 nmol/mg/min, respectively. Of the same mutants in pfAMSDH, N169D also showed the highest activity (22). In other aldehyde dehydrogenases, mutation of the corresponding asparagine to alanine or aspartic acid reduced the activity by approximately 1,000-fold or below detectable limits (27,28).…”

“…(HMSDH) family possesses two conserved arginine residues that are involved in substrate recognition and an isomerization activity (22).…”

“…In initial studies of AMSDH, the identity of the product was inferred based on knowledge of the substrate structure and the catalytic cycle of the dehydrogenation reaction. The in crystallo characterization of the AMSDH reaction revealed that, in addition to oxidation of the aldehyde to its corresponding carboxylic acid, pfAMSDH also isomerizes the 2,3-double bond inside the active site prior to substrate oxidation (22). In the process of trying to determine the conformation of the product of the reaction catalyzed by ALDH8A1, we noticed that the product, 2-AM ( max 330 nm), is unstable and is nonenzymatically bleached in a single kinetic phase with a half-life of 67 min (Fig.…”

“…Crystal structures of the resting enzyme, NAD ϩ -bound complex, ternary complex, catalytic thioacyl and thiohemiacetal intermediates, and several mutants have been reported (21). A hidden isomerase activity of AMSDH has also been uncovered (22). Furthermore, the study of pfAMSDH revealed that, in addition to active-site residues that are broadly conserved across all aldehyde dehydrogenases, the hydroxymuconate semialdehyde dehydrogenase Scheme 1.…”

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

“…It has been demonstrated that embedding an enzyme in a crystalline lattice could dramatically alter reaction kinetics, including extradiol dioxygenases and other enzymes related to HAO in the same metabolic pathway (29)(30)(31)(32)(33). Here, we first tested the catalytic competency of single crystals of HAO using a benchtop spectrophotometer.…”