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.

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.

A Genetic Screen To Identify Genes Influencing the Secondary Redox Couple NADPH/NADP+ in the Yeast Saccharomyces cerevisiae

NADPH is an important cofactor in the cell. In addition to its role in the biosynthesis of critical metabolites, it plays crucial roles in the regeneration of the reduced forms of glutathione, thioredoxins and peroxiredoxins. The enzymes and pathways that regulate NADPH are thus extremely important to understand, and yet are only partially understood. We have been interested in understanding how NADPH fluxes are altered in the cell. We describe here both an assay and a genetic screen that allows one to discern changes in NADPH levels. The screen exploits the secondary redox property of NADPH. At low levels of glutathione we show that the redox contributions of NADPH become critical for growth, and we have used this to develop a genetic screen for genes affecting NADPH homeostasis. The screen was validated in pathways that both directly (pentose phosphate pathway) and indirectly (glycolytic pathway) affect NADPH levels, and was then exploited to identify mitochondrial genes that affect NADPH homeostasis. A total of 239 mitochondrial gene knockouts were assayed using this screen. Among these, several genes were predicted to play a role in NADPH homeostasis. This included several new genes of unknown function, and others of poorly defined function. We examined two of these genes, FMP40 which encodes a protein required during oxidative stress and GOR1, glyoxylate reductase. Our studies throw new light on these proteins that appear to be major consumers of NADPH in the cell. The genetic screen is thus predicted to be an exceedingly useful tool for investigating NADPH homeostasis.

Glyoxylate reductase as versatile enzymatic system for pharmaceutical and medical use

The chiral homogeneity of a chemical compound is the main prerequisite in safety and efficiency of drug substances and generation of single enantiomers of drug intermediates in pharmaceutical industry. Over the past several years there have been an increase wide variety of enzymes and bioengineered microorganisms used for biotransformation of chemicals with chemo- regio- and enantioselectivity. The direct evolution – a combination of biochemistry, molecular biology, structural biology and bioinformatics predictions can modulate enzyme stability, reactivity or substrate specificity. One example of enzymes used in pharmaceutical industry are enzymes from the D-2-hydroxyacid dehydrogenase (2HADH) family. They catalyze reversible reduction of 2-oxoacids to 2-hydroxyacids in an NAD(P)H dependent manner, playing a key role in metabolism of many organisms. One of the enzyme group from 2HADH family is glyoxylate reductase (GR). The advantages of these enzymes have been recently recognized by pharmaceutical and biotechnological industry, considering a possibility of highly stereospecific biotransformation of α-ketoacids into homochiral α-hydroxyacids, as important industrial intermediates. The glycolic acid, reduced by the enzyme into ethylene glycol, the smallest member of the α-hydroxy acid family, is nowadays obtained in a large-scale industrial process. The monomeric structure of the enzyme comprises two domains typical for NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). Several crystal X-ray structures of glyoxylate reductase from different species have been already solved, but only few of them were determined with the bound substrate and/or cofactor. Such structures are crucial in understanding the reaction mechanism and for predicting or designing the structures of new substrates for the enzyme. We will communicate the structural study of two homodimeric glyoxylate reductases from S. meliloti (SmGR1 and SmGR2). We have solved the crystal structures for SmGR1 and SmGR2 with bound oxalate and NADPH and compared them with the structures of other glyoxylate reductases subfamily members: from Rhizobium etli (with L(+)-tartaric acid and NADP) and human (with (2 R )-2,3-dihydroxypropanoic acid and NADPH). During the ligand and cofactor binding the catalytic domain rotates towards coenzyme-binding site, changing its structure from open to close conformation. Using biochemical tools and kinetics approaches we have also shown that Sm GR1 and Sm GR2 possess substrate specificity to hydroxypuryvate, hydroxylphenylpyruvate and gloxylate, providing new insights into the potential pharmaceutical and medical uses of this family of enzymes.

Glyoxylate reductase as versatile enzymatic system for pharmaceutical and medical use

The chiral homogeneity of a chemical compound is the main prerequisite in safety and efficiency of drug substances and generation of single enantiomers of drug intermediates in pharmaceutical industry. Over the past several years there have been an increase wide variety of enzymes and bioengineered microorganisms used for biotransformation of chemicals with chemo- regio- and enantioselectivity. The direct evolution – a combination of biochemistry, molecular biology, structural biology and bioinformatics predictions can modulate enzyme stability, reactivity or substrate specificity. One example of enzymes used in pharmaceutical industry are enzymes from the D-2-hydroxyacid dehydrogenase (2HADH) family. They catalyze reversible reduction of 2-oxoacids to 2-hydroxyacids in an NAD(P)H dependent manner, playing a key role in metabolism of many organisms. One of the enzyme group from 2HADH family is glyoxylate reductase (GR). The advantages of these enzymes have been recently recognized by pharmaceutical and biotechnological industry, considering a possibility of highly stereospecific biotransformation of α-ketoacids into homochiral α-hydroxyacids, as important industrial intermediates. The glycolic acid, reduced by the enzyme into ethylene glycol, the smallest member of the α-hydroxy acid family, is nowadays obtained in a large-scale industrial process. The monomeric structure of the enzyme comprises two domains typical for NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). Several crystal X-ray structures of glyoxylate reductase from different species have been already solved, but only few of them were determined with the bound substrate and/or cofactor. Such structures are crucial in understanding the reaction mechanism and for predicting or designing the structures of new substrates for the enzyme. We will communicate the structural study of two homodimeric glyoxylate reductases from S. meliloti (SmGR1 and SmGR2). We have solved the crystal structures for SmGR1 and SmGR2 with bound oxalate and NADPH and compared them with the structures of other glyoxylate reductases subfamily members: from Rhizobium etli (with L(+)-tartaric acid and NADP) and human (with (2 R )-2,3-dihydroxypropanoic acid and NADPH). During the ligand and cofactor binding the catalytic domain rotates towards coenzyme-binding site, changing its structure from open to close conformation. Using biochemical tools and kinetics approaches we have also shown that Sm GR1 and Sm GR2 possess substrate specificity to hydroxypuryvate, hydroxylphenylpyruvate and gloxylate, providing new insights into the potential pharmaceutical and medical uses of this family of enzymes.