Identification and Characterization of Genes Encoding the Hydroxypyruvate Reductases in Chlamydomonas Reveal Their Distinct Roles in Photorespiration

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms, such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a photorespiration system different from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductases in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together, we identify five proteins to be good candidates for CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a nicotinamide adenine dinucleotide (NADH)-dependent enzyme in mitochondria, may function as the major component of photorespiration. Its deletion causes severe photorespiratory defects. CrHPR2 takes part in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, the results reveal that CrHPRs are far more complex than previously recognized and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate both modification of photorespiration and genetic engineering for crop improvement by synthetic biology.

Metabolite Profiling in Arabidopsis thaliana with Moderately Impaired Photorespiration Reveals Novel Metabolic Links and Compensatory Mechanisms of Photorespiration

Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsis thaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as 13C- and 14C-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism.

Integrative analysis of the hydroxypyruvate reductases revealing their distinct roles in photorespiration of Chlamydomonas

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a different photorespiration system from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductase in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together we identify five proteins to be good candidates as CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a NADH-dependent enzyme in mitochondria, may function as the major component of photorespiration, and deletion of CrHPR1 causes severe photorespiratory defects. CrHPR2 takes parts in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, our results reveal that the CrHPRs are far more complex than previously recognized, and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate the genetic engineering for crop improvement by synthetic biology.

Production of Ethylene Glycol from Glycerol Using an In Vitro Enzymatic Cascade

Glycerol is a readily available and inexpensive substance that is mostly generated during biofuel production processes. In order to ensure the viability of the biofuel industry, it is essential to develop complementing technologies for the resource utilization of glycerol. Ethylene glycol is a two-carbon organic chemical with multiple applications and a huge market. In this study, an artificial enzymatic cascade comprised alditol oxidase, catalase, glyoxylate/hydroxypyruvate reductase, pyruvate decarboxylase and lactaldehyde:propanediol oxidoreductase was developed for the production of ethylene glycol from glycerol. The reduced nicotinamide adenine dinucleotide (NADH) generated during the dehydrogenation of the glycerol oxidation product d-glycerate can be as the reductant to support the ethylene glycol production. Using this in vitro synthetic system with self-sufficient NADH recycling, 7.64 ± 0.15 mM ethylene glycol was produced from 10 mM glycerol in 10 h, with a high yield of 0.515 ± 0.1 g/g. The in vitro enzymatic cascade is not only a promising alternative for the generation of ethylene glycol but also a successful example of the value-added utilization of glycerol.

Pathophysiology and Treatment of Hyperoxaluria

Humans cannot degrade oxalate. Thus, oxalate that is generated in the liver and/or absorbed from the intestine must be eliminated by the kidneys. Among genetic causes, primary hyperoxaluria (PH) type 1 is the most common and occurs due to deficiency of hepatic peroxisomal alanine glyoxalate aminotransferase. PH2 is caused by deficiency of lysosomal glyoxalate reductase or hydroxypyruvate reductase, whereas PH3 results from deficiency of mitochondrial 4-hydroxy-2-oxoglutarate aldolase. Enteric hyperoxaluria is caused by excessive colonic oxalate absorption due to any type of fat malabsorption. The diagnosis of hyperoxaluria is based on the history, 24-hour urine studies, and genetic testing. Early diagnosis and timely intervention are essential. To treat PH, adequate fluid intake, inhibitors of calcium oxalate crystallization (citrate or neutral phosphorus), and pyridoxine-in responsive patients are all important. Intensive dialysis and prompt kidney or combined kidney-liver transplantation are essential to minimize systemic oxalosis if renal failure occurs. Dietary modifications (low fat, low oxalate, and adequate calcium) are key for enteric hyperoxaluria. Calcium can be used as an oxalate binder. Newer modalities including oxalate degrading bacteria, oral oxalate decarboxylase preparations, and inhibitory ribonucleic acids are all under investigation. This review contains 9 figures, 6 tables, and 90 references. Key Words: bariatric surgery, calcium oxalate, dialysis, enteric hyperoxaluria, fat malabsorption, genetic testing, kidney stone, nephrolithiasis, oxalate, oxalate decarboxylase, Oxalobacter formigenes, primary hyperoxaluria, pyridoxine, transplantation, urolithiasis