Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine,Arginine, and Lysine Residues

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.     

Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone

Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.     

Enzymatic metabolism of 3-deoxyglucosone,a Maillard intermediate

Summary In order to verify the formation of endogenous 3-deoxyglucosone (3-DG), an intermediate compound in the Maillard reaction, we tried to detect 3-deoxyfructose (3-DF) which is main metabolite of 3-DG. Endogenous 3-DF was detected in the urine of normal and diabetic rats by the oral administration of 3-DG-free feed. Metabolizing activities of crude extracts prepared from porcine organs were examined using methylglyoxal (MG) and 3-DG as substrates. NAD- or NADP-dependent 2-oxoaldehyde dehydrogenase activity was detected in liver, kidney, small intestine and lung. On the other hand, NADH- or NADPH-dependent 2-oxoaldehyde reductase activity was detected in all porcine organs in which liver and kidney contained higher activity of NADPH-dependent enzyme than the other organs. The reductase which catalyzes the reduction of 3-DG to 3-DF and MG to acetol, was purified and characterized from porcine kidney. The enzyme was the same to NADPH-dependent-2-oxoaldehyde reductase from porcine liver, which is speculated to prevent the advanced stage of the Maillard reaction as a self-defense enzyme.     

Antioxidant and pro-oxidant actions of resveratrol on human serum albumin in the presence of toxic diabetes metabolites: Glyoxal and methyl-glyoxal

Methylglyoxal (MGO) and glyoxal (GO) are attracting considerable attention because of their role in the onset of diabetes symptoms. Therefore, to comprehend the molecular fundamentals of their pathological actions is of the utmost importance. In this study, the molecular interactions between resveratrol (RES) and human serum albumin (HSA) and the ability of the stilbene to counteract the oxidative damage caused by pathological concentrations of MGO and GO to the human plasma protein, was assessed. The oxidation of Cys34 in HSA as well as the formation of specific protein semialdehydes AAS (α-aminoadipic), GGS (γ-glutamic) and the accumulation of Advanced Glycation End-products (AGEs) was investigated. Resveratrol was found to neutralize both α-dicarbonyls by forming adducts detected by HESI-Orbitrap-MS. This antioxidant action was manifested in a significant reduction of AGEs. However, RES-α-dicarbonyl conjugates oxidized Cys34 and lysine, arginine and/or proline by a nucleophilic attack on SH and ε-NH groups in HSA. The formation of specific semialdehydes in HSA after incubation with GO and MGO at pathological concentrations was reported for the first time in this study, and may be used as early and specific biomarkers of the oxidative stress undergone by diabetic patients. The pro-oxidative role of the RES-α-dicarbonyl conjugates should be further investigated to clarify whether this action leads to positive or harmful clinical consequences. The biological relevance of human protein carbonylation as a redox signaling mechanism and/or as a reflection of oxidative damage and disease should also be studied in future works.     

Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases

Pure chiral molecules are needed in the pharmaceutical and chemical industry as intermediates for the production of drugs or fine chemicals. Microorganisms represent an attractive alternative to chemical synthesis since they have the potential to generate single stereoisomers in high enantiomeric excess (ee). The baker's yeast Saccharomyces cerevisiae can notably reduce dicarbonyl compounds (in particular alpha- and beta-diketones and keto esters) to chiral alcohols with high ee. However, products are formed at a low rate. Moreover, large amounts of co-substrate are required for the regeneration of NADPH that is the preferred co-factor in almost all the known dicarbonyl reductions. Traditionally, better ee, reduction rate and product titre have been achieved via process engineering. The advent of recombinant DNA technology provides an alternative strategy to improve productivity and yield by strain engineering. This review discusses two aspects of strain engineering: (i) the generation of strains with higher reductase activity towards dicarbonyl compounds and (ii) the optimisation of co-substrate utilisation for NADPH cofactor regeneration.     

Metabolism of 3-deoxyglucosone,an intermediate compound in the Maillard reaction,administered orally or intravenously to rats

Tha Amadori rearrangement compound, the product in the early step of the Maillard reaction of proteins with glucose, is known to be degraded into 3-deoxyglucosone (3DG), a 2-oxoaldehyde. In order to elucidate the metabolic pathway of 3DG, [¹⁴C]3DG was synthesized from [¹⁴C]-glucose and administered to rats orally and intravenously. 2 h after oral administration of [¹⁴C]3DG, the percentages of radioactivity (RaI%) in stomach, small intestine and urine were 3.9, 60 and 6.4%, respectively, while RaI% in liver, kidney, spleen, blood and CO₂ were less than 0.5%. The absorption rate of 3DG was obviously lower in comparison with that of glucose. 3 h after intravenous administration of [¹⁴C]3DG, the RaI% in urine was 72% and those in liver, kidney, spleen, blood and CO₂ were less than 1%. It therefore appeared that the absorbed 3DG was not biologically utilized by the rats, but was rapidly excreted in the urine. Some metabolites of [¹⁴C]3DG were detected in urine by TLC-autoradiography. The main metabolite was purified and identified as 3-deoxyfructose by FD-MS and ¹³C-NMR spectroscopy, indicating that the aldehyde group of 3DG was reduced to an alcohol.     

α,β-dicarbonyl reduction by Saccharomycesd-arabinose dehydrogenase

An α,β-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme d-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive α,β-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent K_m values of ∼ 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s^− 1 at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP⁺ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of α,β-dicarbonyl substrates was further supported by the observation that ara1-Δ knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.