Carbonyl Metabolism Research Articles

Genetic variants as modulators of human (patho) physiology

The neurotransmitter dopamine (DA) is important for numerous biological functions, including control of movement. Oxidation of DA to highly toxic and reactive species has been hypothesized to contribute to the selective neurodegeneration observed in Parkinson's disease (PD). DA catabolism is initiated by oxidative deamination via monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL). Such metabolism can be problematic as it greatly increases the toxicity of DA by production of DOPAL, known to be a toxic and reactive intermediate. DOPAL undergoes carbonyl metabolism primarily via aldehyde dehydrogenase (ALDH) enzymes to a less toxic acid product. Previous studies from our laboratory have shown that cellular ALDH enzymes are sensitive towards products of oxidative stress and lipid peroxidation, which are thought to be elevated during PD pathogenesis. Inhibition of ALDH and the resulting accumulation of DOPAL are concerning as DOPAL is toxic to dopaminergic cells, readily modifies proteins and causes protein aggregation. In addition, pesticides with association between exposure and PD incidence can interfere with DA metabolism and trafficking and/or ALDH activity, directly or indirectly, yielding elevation of DOPAL. Therefore, impairment of carbonyl metabolism is a potential mechanistic link between cellular insult and generation of a toxic and reactive intermediate endogenous to dopamine neurons.

Annotation of 1350 Common Genetic Variants of the 19 ALDH Multigene Family from Global Human Genome Aggregation Database (gnomAD).

Human aldehyde dehydrogenase (ALDH) is a multigene family with 19 functional members encoding a class of diverse but important enzymes for detoxification or biotransformation of different endogenous and exogenous aldehyde substrates. Genetic mutations in the ALDH genes can cause the accumulation of toxic aldehydes and abnormal carbonyl metabolism and serious human pathologies. However, the physiological functions and substrate specificity of many ALDH genes are still unknown. Although many genetic variants of the ALDH gene family exist in human populations, their phenotype or clinical consequences have not been determined. Using the most comprehensive global human Genome Aggregation Database, gnomAD, we annotated here 1350 common variants in the 19 ALDH genes. These 1350 common variants represent all known genetic polymorphisms with a variant allele frequency of ≥0.1% (or an expected occurrence of ≥1 carrier per 500 individuals) in any of the seven major ethnic groups recorded by gnomAD. We detailed 13 types of DNA sequence variants, their genomic positions, SNP ID numbers, and allele frequencies among the seven major ethnic groups worldwide for each of the 19 ALDH genes. For the 313 missense variants identified in the gnomAD, we used two software algorithms, Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant From Tolerant (SIFT), to predict the consequences of the variants on the structure and function of the enzyme. Finally, gene constraint analysis was used to predict how well genetic mutations were tolerated by selection forces for each of the ALDH genes in humans. Based on the ratio of observed and expected variant numbers in gnomAD, the three ALDH1A gene members, ALDH1A1, ALDH1A2, and ALDH1A3, appeared to have the lowest tolerance for loss-of-function mutations as compared to the other ALDH genes (# observed/# expected ratio 0.15–0.26). These analyses suggest that the ALDH1A1, ALDH1A2, and ALDH1A3 enzymes may serve a more essential function as compared with the other ALDH enzymes; functional loss mutations are much less common in healthy human populations than expected. This informatic analysis may assist the research community in determining the physiological function of ALDH isozymes and associate common variants with clinical phenotypes.

Enhanced Catecholamine Flux and Impaired Carbonyl Metabolism Disrupt Cardiac Mitochondrial Oxidative Phosphorylation in Diabetes Patients.

Aims: Catecholamine metabolism via monoamine oxidase (MAO) contributes to cardiac injury in models of ischemia and diabetes, but the pathogenic mechanisms involved are unclear. MAO deaminates norepinephrine (NE) and dopamine to produce H2O2 and highly reactive "catecholaldehydes," which may be toxic to mitochondria due to the localization of MAO to the outer mitochondrial membrane. We performed a comprehensive analysis of catecholamine metabolism and its impact on mitochondrial energetics in atrial myocardium obtained from patients with and without type 2 diabetes. Results: Content and maximal activity of MAO-A and MAO-B were higher in the myocardium of patients with diabetes and they were associated with body mass index. Metabolomic analysis of atrial tissue from these patients showed decreased catecholamine levels in the myocardium, supporting an increased flux through MAOs. Catecholaldehyde-modified protein adducts were more abundant in myocardial tissue extracts from patients with diabetes and were confirmed to be MAO dependent. NE treatment suppressed mitochondrial ATP production in permeabilized myofibers from patients with diabetes in an MAO-dependent manner. Aldehyde dehydrogenase (ALDH) activity was substantially decreased in atrial myocardium from these patients, and metabolomics confirmed lower levels of ALDH-catalyzed catecholamine metabolites. Proteomic analysis of catechol-modified proteins in isolated cardiac mitochondria from these patients identified >300 mitochondrial proteins to be potential targets of these unique carbonyls. Innovation and Conclusion: These findings illustrate a unique form of carbonyl toxicity driven by MAO-mediated metabolism of catecholamines, and they reveal pathogenic factors underlying cardiometabolic disease. Importantly, they suggest that pharmacotherapies targeting aldehyde stress and catecholamine metabolism in heart may be beneficial in patients with diabetes and cardiac disease. Antioxid. Redox Signal. 35, 235-251.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous ‘ene’-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

MDR-alcohol dehydrogenases

Close to 80 years of research have brought MDR alcohol dehydrogenases (ADHs) from unknown molecular concepts to molecules known in exact detail regarding structural, functional and evolutionary properties. They can be traced backwards in at least six stages of development, to essentially the origin of cellular life, and have been monitored in a long series of biannual meetings on “Carbonyl Metabolism”. In between each of these latest meetings, a roughly three-fold increase in known totals of MDR databank entries has been apparent, bringing the total now of known MDR-ADH entries to approaching half a million forms.

Aldehyde dehydrogenase inhibition generates a reactive dopamine metabolite autotoxic to dopamine neurons

The neurotransmitter dopamine (DA) is important for numerous biological functions, including control of movement. Oxidation of DA to highly toxic and reactive species has been hypothesized to contribute to the selective neurodegeneration observed in Parkinson's disease (PD). DA catabolism is initiated by oxidative deamination via monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL). Such metabolism can be problematic as it greatly increases the toxicity of DA by production of DOPAL, known to be a toxic and reactive intermediate. DOPAL undergoes carbonyl metabolism primarily via aldehyde dehydrogenase (ALDH) enzymes to a less toxic acid product. Previous studies from our laboratory have shown that cellular ALDH enzymes are sensitive towards products of oxidative stress and lipid peroxidation, which are thought to be elevated during PD pathogenesis. Inhibition of ALDH and the resulting accumulation of DOPAL are concerning as DOPAL is toxic to dopaminergic cells, readily modifies proteins and causes protein aggregation. In addition, pesticides with association between exposure and PD incidence can interfere with DA metabolism and trafficking and/or ALDH activity, directly or indirectly, yielding elevation of DOPAL. Therefore, impairment of carbonyl metabolism is a potential mechanistic link between cellular insult and generation of a toxic and reactive intermediate endogenous to dopamine neurons.

16th Carbonyl Metabolism Meeting: from enzymology to genomics

The 16th International Meeting on the Enzymology and Molecular Biology of Carbonyl Metabolism, Castle of Ploen (Schleswig-Holstein, Germany), July 10–15, 2012, covered all aspects of NAD(P)-dependent oxido-reductases that are involved in the general metabolism of xenobiotic and physiological carbonyl compounds. Starting 30 years ago with enzyme purification, structure elucidation and enzyme kinetics, the Carbonyl Society members have meanwhile established internationally recognized enzyme nomenclature systems and now consider aspects of enzyme genomics and enzyme evolution along with their roles in diseases. The 16th international meeting included lectures from international speakers from all over the world.

Metabolism of SO2 binding compounds by Oenococcus oeni during and after malolactic fermentation in white wine

Sulfur dioxide (SO2) is the key additive for the preservation of wines. Carbonyl and keto compounds in wine can bind to SO2 and decrease its efficacy, resulting in higher total SO2 requirements. Increased consumer demand for low sulfite and organic wines pose production challenges if SO2 binders have not been properly managed during vinification. Malolactic fermentation (MLF) has been known to reduce bound SO2 levels but detailed time course studies are not available. In this work, the kinetics of major SO2 binding compounds and malic acid were followed during MLF in wine with 12 commercially available strains of Oenococcus oeni. Pyruvic acid, acetaldehyde and α-ketoglutaric acid were degraded to various degrees by O. oeni, but galacturonic acid was not. At the time of malic acid depletion, percent degradation of pyruvate, α-ketoglutaric acid and acetaldehyde was 49%, 14% and 30%, respectively. During MLF, the decrease in average bound SO2 levels, as calculated from carbonyl metabolism, was 22%. The largest reduction in wine carbonyl content occurred in the week after completion of MLF and was 53% (107mg/L to 34mg/L) calculated as bound SO2. Prolonged activity of bacteria in the wines (up to 3weeks post malic acid depletion) resulted only in reduced additional reductions in bound SO2 levels.The results suggest that microbiological wine stabilization one week after malic acid depletion is an effective strategy for maximum removal of SO2 binders while reducing the risk of possible post-ML spoilage by O. oeni leading to the production acetic acid and biogenic amines.

The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site

Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group reduction in alpha-dicarbonyls differs from that utilized in the conversion of xylose.

Characterization of human DHRS4: An inducible short-chain dehydrogenase/reductase enzyme with 3β-hydroxysteroid dehydrogenase activity

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27 kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and α-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all- trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C 19/C 21-steroids into 3β-hydroxysteroids. The stereospecific conversion to 3β-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor α ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3β-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

Catalytic mechanism and substrate selectivity of aldo-keto reductases: Insights from structure-function studies of Candida tenuis xylose reductase

Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the epsilon-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications.

Transgenic Mice Over-expressing Dicarbonyl/L-xylulose Reductase Gene Crossed with KK-Ay Diabetic Model Mice: An Animal Model for the Metabolism of Renal Carbonyl Compounds

Carbonyl compounds in the blood stream tend to accumulate in the kidney of diabetic or end stage renal failure subjects. Previously we isolated cDNA encoding dicarbonyl/L-xylulose reductase (DCXR) from a mouse kidney cDNA library. In the present study, transgenic (Tg) mice were generated to study the functional role of DCXR in the kidney. With a six-fold increase in the DCXR protein expression levels in the kidney, the homozygous Tg mice did not show any notable histological abnormalities. While the elevated DCXR expression was observed throughout the body, its renal distribution was similar to that of the endogenous DCXR protein, namely, the major expression site was the collecting tubules, along with moderate expression in other tubules and Bowman's capsule, but it was absent from the interstitial area and glomeruli. The Tg mice were crossed with KK-A(y) diabetic model mice to examine the role of DCXR in the progression of diabetic nephropathy. The resulting progeny, Tg/A(y), showed lighter body weight, lower levels of blood glucose, water uptake and creatinine clearance compared to their +/A(y) littermates. Although remarkable pathological differences were not observed at the microscopic level and in the renal accumulation of carboxymethyl lysine, the data imply that DCXR might function in the metabolism of glucose or carbonyl compounds, and play a protective role in a kidney which is under hyperglycemic pressure. The DCXR Tg mice and the Tg x KK-A(y) hybrid mice, therefore, serve as specific models for carbonyl metabolism in the kidney with diabetic background.

Human Carbonyl Reduction Pathways and a Strategy for Their Study In Vitro

Carbonyl reduction plays a significant role in physiological processes throughout the body. Although much is known about endogenous carbonyl metabolism, much less is known about the roles of carbonyl‐reducing enzymes in xenobiotic metabolism. Multiple pathways exist in humans for metabolizing carbonyl moieties of xenobiotics to their corresponding alcohols, readying these molecules for subsequent conjugation and/or excretion. When exploring carbonyl reduction clearance pathways for a drug development candidate, it is possible to assess the relative contributions of these enzymes due to their differences in subcellular locations, cofactor dependence, and inhibitor profiles. In addition, the contributions of these enzymes may be explored by varying incubation conditions, such as pH. Presently, individual isoforms of carbonyl‐reducing enzymes are not widely available, either in recombinant or purified form. However, it is possible to study carbonyl reduction clearance pathways from simple experiments with commercially available reagents. This article provides an overview of carbonyl‐reducing enzymes, including some kinetic data for substrates and inhibitors. In addition, an experimental strategy for the study of these enzymes in vitro is presented.

Expression of rat aldehyde reductase AKR7A1: influence of age and sex and tissue-specific inducibility☆ 1

The regulation of the aldo-keto reductase AKR7A1 was examined in the livers of male and female rats during development by using Western blots, and its contribution to carbonyl metabolism was assessed by using enzyme assays. Hepatic levels of AKR7A1 are low in fetal rats and rise to a peak at around 6 weeks of age in animals of both sexes. Higher levels of the enzyme are found in adult male rat liver than in adult female rat liver. The reductase, therefore, appears to be subject to sex-specific regulation. The effect of growth hormone in mediating this difference in expression was examined by using hypophysectomized animals whose serum growth hormone levels had been feminized by continuous administration. Results demonstrate that such treatment leads to a reduction in AKR7A1 expression. AKR7A1 was found to be constitutively expressed in rat tissues such as liver, kidney, small intestine, and testis, but it was not detected in nasal mucosa, skeletal muscle, heart, adrenal gland, brain, or spleen. However, AKR7A1 was inducible by the synthetic antioxidant ethoxyquin in liver, kidney, and small intestine, but not in the other tissues examined. These results show that levels of this important detoxication enzyme vary considerably according to age and sex and that dietary antioxidants can also influence its level in several tissues.

Enzymology and molecular biology of carbonyl metabolism 5, advances in experimental medicine and biology : vol 372, edited by H Weiner, RS Holmes, B Wermut, Plenum Press, NY and London, 1995, 442 p

Although obesity represents a potential public health crisis, our understanding of public perceptions of obesity, emotional responses to the obese, and related policy preferences is limited.We employed Weiner's attribution theory of controllability (Weiner, 1988, 2011) to examine perceived causes of obesity, emotional responses, and related policy implications. If the perceived cause is controllable (eating and lifestyle habits), we expected less sympathy and greater anger toward obese people and support for prejudicial hiring policies based on weight. If the cause is perceived as uncontrollable (genetic), sympathy is anticipated as well as opposition to such hiring policies.We conducted multivariate analyses with data from two nationally representative surveys of U.S. adults.Our findings supported the hypotheses, showing that sympathy and lack of anger toward obese people are strongly determined by a genetic attribution for obesity. Moreover, sympathy and the genetic attribution are significant predictors of opposition to hiring policies that discriminate against obese people. Finally, in a second study, chief among several causal attributions for obesity—lack of will power, lack of exercise, marketing of foods, and genetics—we discovered biological attribution functions as the key predictor of perceptions that obese people are frequent targets of discrimination. We suggest governments and the food industry should be more attuned to the underlying beliefs of the public about the causes of obesity when formulating programs and policies to address the issue.

Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts

Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/ hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism. Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3α-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate. The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors. In all fractions tested, steroids of the androstane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction. Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossreactions in all fractions tested, even in those of the insect and the procaryont. A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3α-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggest the existence of structural homologies within these carbonyl reducing enzymes. This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen. These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.

BOOK REVIEWS

Journal Article BOOK REVIEWS Get access Enzymology and Molecular Biology of Carbonyl Metabolism. 2. Aldehyde Dehydrogenase, Alcohol Dehydrogenase and Aldo-keto Reductase . Edited by H. Weiner and T. G. Flynn , Alan R. Liss , New York , 1989 , 432 pp. TIMOTHY J. PETERS TIMOTHY J. PETERS Search for other works by this author on: Oxford Academic PubMed Google Scholar Alcohol and Alcoholism, Volume 25, Issue 4, 1990, Page 439, https://doi.org/10.1093/oxfordjournals.alcalc.a045032 Published: 01 July 1990

BOOK REVIEWS

BOOK REVIEWS Enzymology and Molecular Biology of Carbonyl Metabolism: Aldehyde Dehydrogenase Aldo-Keto Reductase and Alcohol Dehydrogenase . Edited by Henry Weiner and T. Geoffrey Flynn . Alan R. Liss Inc ., New York , 1987 , 447 pp., £50.00. ABDULLA A.-B. BADAWY ABDULLA A.-B. BADAWY Search for other works by this author on: Oxford Academic PubMed Google Scholar Alcohol and Alcoholism, Volume 24, Issue 4, 1989, Page 361, https://doi.org/10.1093/oxfordjournals.alcalc.a044927 Published: 01 July 1989

Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog.

Cloned cDNA sequences specific for prostaglandin F (PGF) synthase have been isolated from a cDNA library of bovine lung mRNA sequences. Nucleotide-sequence analyses of cloned cDNA inserts have revealed that PGF synthase consists of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a Mr of 36,666. The sequence analysis indicates that bovine lung PGF synthase shows 62% identical plus conservative substitutions compared with human liver aldehyde reductase [Wermuth, B., Omar, A., Forster, A., Francesco, C., Wolf, M., Wartburg, J.P., Bullock, B. & Gabbay, K.H. (1987) in Enzymology and Molecular Biology of Carbonyl Metabolism: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase, eds. Weiner, H. & Flynn, T.G. (Liss, New York), pp. 297-307], which is similar to PGF synthase in molecular weight and substrate specificity. However, comparison of the amino acid sequence of PGF synthase with the National Biomedical Research Foundation protein data base reveals that the sequences of 225 amino acids from C termini of epsilon-crystallin of the European common frog (Rana temporaria) [Tomarev, S.I., Zinovieva, R.D., Dolgilevich, S.M., Luchin, S.V., Krayev, A.S., Skryabin, K.G. & Gause, G.G. (1984) FEBS Lett. 171, 297-302] and of PGF synthase show 77% identical and conservative substitutions without deletions/additions. The result suggests that European common frog lens epsilon-crystallin is identical to bovine lung PGF synthase.

Immunization for Feline Panleukopenia

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous ‘ene’-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.