Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite''s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD⁺). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD⁺ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD⁺ metabolism in these parasites.     

Towards catalyst compartimentation in combined chemo- and biocatalytic processes: Immobilization of alcohol dehydrogenases for the diastereoselective reduction of a β-hydroxy ketone obtained from an organocatalytic aldol reaction

The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with “free” enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor.     

Cytosolic NAD content strictly depends on ATP concentration in isolated liver cells

By focusing on the question of the thermodynamic relationships involved in the regulation of biological energy conversion, bioenergetic studies usually consider the free pyridine and adenine nucleotide rather than their total pools, in either cytosol or mitochondria. In this study, we report a new observation that, at steady state, nicotinamide nucleotide content is increased by a rise in the ATP content of the whole cell under physiological conditions. It is a straight line relationship when only NAD⁺ and ATP are considered. When regarding the compartmentation of this phenomenon, it appears that the linear relationship between [NAD⁺] and [ATP] occurs only in the cytosol. Such a dependence could be a supplementary mechanism of regulation between various metabolic pathways in the liver cell.     

NADP-Dependent enzymes. II: Evolution of the mono- and dinucleotide binding domains

Nicotinamide adenine dinucleotides [NAD and NADP with both referred to as NAD(P)] are among the more diffuse redox cofactors. Despite their stereochemical similarity where the only difference is a phosphomonoester on the ribose near the adenine of NADP, they show different biochemical reactivities with NAD behaving as an oxidant and NADP as a reductant. NAD(P)-dependent enzymes generally share a common open α/β fold with few exceptions only recently structurally characterized. This study of the molecular evolution of the NAD(P) binding domains, possible given the large number of known molecular structures, addresses two main questions: 1) can a common fold exist in different biological systems (divergent evolution) and 2) does a relationship exist among similar biological systems that display different folds (convergent evolution)? Both the structures of mono- and dinucleotide binding domains have been classified by cluster analysis based on the similarity evaluated by their main chain Cα superposition. Moreover, the cofactor conformations and the stereochemical characteristics of their pockets have also been classified by analogous methods on the basis of the published tertiary structures. Two primary results appear: 1) the classification of the mononucleotide binding domains is different from that of the dinucleotide binding folds and 2) both divergent and convergent evolutionary pathways can be hypothesized, the latter less frequently observed and less pronounced but nevertheless evident. The generally accepted hypothesis that dinucleotide binding domains have evolved by gene duplication of primordial genes coding for the smaller mononucleotide binding domains is acceptable but the two halves of the resulting dinucleotide binding domains are evolutionarly uncorrelated. The NH₂-terminal mononucleotide binding domain is less variable than the COOH-terminal half, probably because it involves the binding of the ADP moiety of NAD(P) invariant in all examined systems. There is evidence to postulate that evolutionary pathways for NAD(P)-dependent enzymes are both divergent and convergent. In fact, nearly all combinations of similarity/dissimilarity in overall fold, cofactor conformation, and cofactor binding pocket structural characteristics for each enzyme pair examined are possible. The NAD(P)-dependent enzymes apparently provide a canonical example of an evolutionary principle that “anything goes.” © 1997 Wiley-Liss Inc.     

Biofuel anode based on d-glucose dehydrogenase,nicotinamide adenine dinucleotide and a modified electrode

A biofuel cell anode has been made from a modified graphite electrode and immobilized d-glucose dehydrogenase [β-d-glucose:NAD(P)⁺ 1-oxidoreductase, EC 1.1.1.4 7] so that energy could be drawn from the conversion of d-glucose to d-gluconic acid. An equivalent amount of dihydronicotinamide adenine dinucleotide (NADH) was formed from NAD⁺ and reduced the surface groups of the modified electrode. Reoxidationn of the latter produced the electrons necessary for a power output from the cell. Electrode modification was made by adsorption of N,N-dimethyl-7-amino 1,2-benzophenoxazinium onto the graphite. A current density of 0.2 mA cm^?2 at a cell voltage of ～0.8 V was obtained for more than 8 h with a simulated oxygen cathode. The internal resistance in the cell, in particular in the separator, appeared to be the main current-limiting factor.     

NAD+ accumulation as a metabolic off switch for orthodox pollen

Terrestrial plant pollen is classified into two categories based on its metabolic status: pollen with low-metabolism are termed “orthodox” and pollen with high-metabolism are termed “recalcitrant.” Nicotinamide adenine dinucleotide (NAD) is crucial for a number of metabolisms in all extant organisms. It has recently been shown that NAD homeostasis plays an important role in a broad range of developmental processes and responses to environment. Recently, a reverse genetic approach shed light on the significance of NAD biosynthesis on pollen fate. In orthodox Arabidopsis pollen, NAD⁺ that was accumulated in excess at dispersal dramatically decreased on rehydration. The lack of a key gene that is involved in NAD biosynthesis compromised the excess accumulation. Moreover, absence of the excess accumulation phenocopied the so-called recalcitrant pollen, as demonstrated by the germination inside anthers and the loss of desiccation tolerance. Upon rehydration, NAD⁺-consuming inhibitors impaired tube germination. Taken together, our results suggest that accumulation of NAD⁺ functions as a physiochemical molecular switch for suspended metabolism and that the decrease of NAD⁺ plays a very important role during transitions in metabolic states. Shifting of the redox state to an oxidizing environment may efficiently control the comprehensive metabolic network underlying the onset of pollen germination.     

Characterization of the complete mitochondrial genome of Diaphania pyloalis (Lepidoptera: Pyralididae)

The complete mitochondrial genome (mitogenome) of Diaphania pyloalis (Lepidoptera: Pyralididae) was determined to be 15,298 bp and has the typical gene organization of mitogenomes from lepidopteran insects. It consists of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. The A + T content of this mitogenome is 80.83% and the AT skew is slightly positive. All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which is initiated by CGA. Only the cox2 gene has an incomplete stop codon consisting of just a T. All the tRNA genes display a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome is 332 bp in length, including several common features found in lepidopteran mitogenomes. Phylogenetic analysis showed that the D. pyloalis is close to Pyralididae.     

Generation,Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other''s NAD supply by providing alternative precursors.     

Characterization of the complete mitochondrial genome of Bombyx mori strain H9 (Lepidoptera: Bombycidae)

The complete mitochondrial genome (mitogenome) of Bombyx mori strain H9 (Lepidoptera: Bombycidae) is 15,670 base pairs (bp) in length, encoding 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The nucleotide composition of the genome is highly A + T biased, accounting for 81.31%, with a slightly positive AT skewness (0.059). The arrangement of 13 PCGs is similar to that of other sequenced lepidopterans. All the PCGs are initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which is proposed by the TTAG sequence as observed in other lepidopterans. Unlike the other PCGs, the cox1 and cytochrome c oxidase subunit 2 (cox2) genes have incomplete stop codons consisting of just a T. All tRNAs have typical structures of insect mitochondrial tRNAs, which is different from other sequenced lepidopterans. The structure of A + T-rich region is similar to that of other sequenced lepidopterans, including non-repetitive sequences, the ATAGA binding domain, a 18 bp poly-T stretch and a poly-A element upstream of transfer RNA M (trnM) gene. Phylogenetic analysis shows that the domesticated silkmoth B. mori originated from the Chinese Bombyx mandarina.     

Oxidation of reduced nicotinamide hypoxanthine dinucleotide by intact rat liver mitochondria

1. The rate of NADH oxidation catalyzed by intact rat liver mitochondria is greatly stimulated in the presence of oxidized nicotinamide hypoxanthine dinucleotide (NHD⁺).2. Mitochondrial oxidation of external NHDH is from 20- to 40-fold more rapid than that of NADH, although these coenzymes are oxidized at similar rates by sonicated mitochondria.3. NADH and NADPH inhibit, while NADP⁺ stimulates NHDH oxidation.4. NHDH oxidation is inhibited by rotenone and CN^?.5. NHDH oxidation is coupled to the phosphorylation of ADP to ATP, yielding P:2e^? ratios approaching 3.6. These studies indicate that external NHDH is oxidized by the intramito-chondrial respiratory chain NADH dehydrogenase and that the inner mitochondrial membrane is significantly more permeable to NHDH than to NADH. Mammalian liver mitochondria have been reported to catalyze the enzymatic deamination of NAD(H) to NHD(H) [Buniatian, H. C. (1970) in Handbook of Neurochemistry (Lajtha, A., ed.), Vol. 3, pp. 399–411, Plenum Press, London and New York; Movcessian, S. G. and Manassian, R. F. (1967) in Problems of Brain Biochemistry, Vol. 3, pp. 53–66, Academic Press, Yerevan], suggesting a metabolic function for the deaminated analogue. It is concluded that this deamination reaction may be operative in a mechanism for the oxidation of cytoplasmic NADH by the respiratory chain.     

Nicotinamide adenine dinucleotides permeate through mitochondrial membranes in human Epstein-Barr virus-transformed lymphocytes

Human cultured cells are widely used for the investigation of respiratory chain disorders. Oxidative properties are generally investigated by means of polarographic studies carried out on detergent-permeabilized cells. By studying the oxidative properties of Epstein-Barr virus-transformed B lymphocytes, we found that the respiration was significantly decreased after 3–4 days of cell culture. Simultaneously, we observed that NAD⁺-dependent oxidations (malate, glutamate, pyruvate) became dependent upon the addition of exogenous NAD⁺. The effect of NAD⁺ was shown to be related to an influx of catalytic amount of NAD⁺ into the mitochondrial matrix. A full ability to oxidize NAD⁺-dependent substrates was restored less than 2 h after a change of the culture medium.These observations suggested: (a) the occurrence of fluxes of catalytic amounts of NAD⁺ through the mitochondrial inner membrane in human cells; (b) an early control of mitochondrial metabolism by matrix NAD⁺ content in cells grown under limiting growth conditions; (c) the possible confusion between complex I deficiency and a decrease content of matrix NAD⁺ when using human cultured cells. (Mol Cell Biochem 115–119, 1997)     

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,Leads to Altered Carbohydrate Metabolism in Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD⁺ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD⁺ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.     

Kinetics of Thermal Inactivation of Lactate Dehydrogenase from Rabbit Muscle

The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381–436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme–substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme–substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme.     

Modulating NAD+ metabolism,from bench to bedside

Discovered in the beginning of the 20^th century, nicotinamide adenine dinucleotide (NAD⁺) has evolved from a simple oxidoreductase cofactor to being an essential cosubstrate for a wide range of regulatory proteins that include the sirtuin family of NAD⁺‐dependent protein deacylases, widely recognized regulators of metabolic function and longevity. Altered NAD⁺ metabolism is associated with aging and many pathological conditions, such as metabolic diseases and disorders of the muscular and neuronal systems. Conversely, increased NAD⁺ levels have shown to be beneficial in a broad spectrum of diseases. Here, we review the fundamental aspects of NAD⁺ biochemistry and metabolism and discuss how boosting NAD⁺ content can help ameliorate mitochondrial homeostasis and as such improve healthspan and lifespan.     

Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD⁺ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.     

Characterization of NADPH–cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera

A complete cDNA encoding the NADPH–cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4 kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis–Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC₅₀ = 1.69 μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC₅₀ value ranged from 15 to 90 μM) was also observed.     

Arabidopsis thaliana nicotinate/nicotinamide mononucleotide adenyltransferase (AtNMNAT) is required for pollen tube growth

While mammals and fungi possess nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) isoforms, Arabidopsis thaliana only contains a single NMNAT gene, AtNMNAT (At5g55810). We analyzed the enzymatic activity of the AtNMNAT-encoded protein to determine the role of AtNMNAT in plant development. AtNMNAT catalyzed the synthesis of nicotinate adenine dinucleotide (NaAD) from nicotinate mononucleotide (NaMN) in the Preiss-Handler-dependent pathway, and of nicotinamide adenine dinucleotide (NAD) from nicotiamide mononucleotide (NMN) in the Preiss-Handler-independent pathway. Prominent AtNMNAT expression was detected in the male gametophyte. Moreover, AtNMNAT expression was spatio-temporally regulated during microspore development and pollen tube growth. Disruption of the AtNMNAT gene (atnmnat mutant) was characterized by a decrease in NAD content in pollen. Cytological examinations revealed that the atnmnat mutant was gametophytically impaired in in vivo and in vitro pollen tube growth. Our results suggest that metabolic fulfillment via the NAD pathway is indispensable for normal pollen growth and subsequent normal seed production.     

NADP-Dependent enzymes. I: Conserved stereochemistry of cofactor binding

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2′-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moeties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design. © 1997 Wiley-Liss Inc.     

In silico prediction of the effects of mutations in the human UDP-galactose 4′-epimerase gene: Towards a predictive framework for type III galactosemia

The enzyme UDP-galactose 4′-epimerase (GALE) catalyses the reversible epimerisation of both UDP-galactose and UDP-N-acetyl-galactosamine. Deficiency of the human enzyme (hGALE) is associated with type III galactosemia. The majority of known mutations in hGALE are missense and private thus making clinical guidance difficult. In this study a bioinformatics approach was employed to analyse the structural effects due to each mutation using both the UDP-glucose and UDP-N-acetylglucosamine bound structures of the wild-type protein. Changes to the enzyme's overall stability, substrate/cofactor binding and propensity to aggregate were also predicted. These predictions were found to be in good agreement with previous in vitro and in vivo studies when data was available and allowed for the differentiation of those mutants that severely impair the enzyme's activity against UDP-galactose. Next this combination of techniques were applied to another twenty-six reported variants from the NCBI dbSNP database that have yet to be studied to predict their effects. This identified p.I14T, p.R184H and p.G302R as likely severely impairing mutations. Although severely impaired mutants were predicted to decrease the protein's stability, overall predicted stability changes only weakly correlated with residual activity against UDP-galactose. This suggests other protein functions such as changes in cofactor and substrate binding may also contribute to the mechanism of impairment. Finally this investigation shows that this combination of different in silico approaches is useful in predicting the effects of mutations and that it could be the basis of an initial prediction of likely clinical severity when new hGALE mutants are discovered.