Stable Isotope-Assisted Untargeted Metabolomics Identifies ALDH1A1-Driven Erythronate Accumulation in Lung Cancer Cells.

Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.

Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

Stabilization and conformational isomerization of the cofactor during the catalysis in hydrolytic ALDHs.

Over the past 15 years, mechanistic and structural aspects were studied extensively for hydrolytic ALDHs. One the most striking feature of nearly all X-ray structures of binary ALDH-NAD(P)(+) complexes is the great conformational flexibility of the NMN moiety of the NAD(P)(+), in particular of the nicotinamide ring. However, the fact that the acylation step is efficient in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans and in other hydrolytic ALDHs implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary complex to allow an efficient and stereospecific hydride transfer. Another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the stabilization of the NMN moiety of the cofactor during the acylation step and to provide structural evidence of conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs. The results presented here will be discussed for their relevance to the two-step catalytic mechanism and from an evolutionary viewpoint.

Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.

One of the most striking features of several X-ray structures of CoA-independent ALDHs (aldehyde dehydrogenases) in complex with NAD(P) is the conformational flexibility of the NMN moiety. However, the fact that the rate of the acylation step is high in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of serine for invariant Thr244 and alanine for Lys178 result in a drastic decrease of the efficiency of hydride transfer which becomes rate-limiting. The crystal structure of the binary complex T244S GAPN-NADP shows that the absence of the beta-methyl group leads to a well-defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild-type. The approximately 0.6-unit increase in pK(app) of the catalytic Cys302 observed in the ternary complex for both mutated GAPNs is likely to be due to a slight difference in positioning of the nicotinamide ring relative to Cys302 with respect to the wild-type ternary complex. Taken together, the data support a critical role of the Thr244 beta-methyl group, held in position through a hydrogen-bond interaction between the Thr244 beta-hydroxy group and the epsilon-amino group of Lys178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis.

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.