Search

Salicylaldehyde dehydrogenase (SALD) catalyses the last reaction in the upper pathway of naphthalene degradation: the oxidation of salicylaldehyde to salicylate. This enzyme has been isolated and studied from a few organisms that belong to the betaproteobacteria and gammaproteobacteria, predominantly Pseudomonas putida. Furthermore, there is only one crystal structure of this enzyme, which was obtained from P. putida G7. Here, crystallographic studies and analysis of the crystal structure of an Alpine soil metagenome-derived SALD (SALDAP) from an alphaproteobacterium are presented. The SALDAP gene was discovered using gene-targeted sequence assembly and it was cloned into a pLATE51 vector. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) cells and the soluble protein was purified to homogeneity. The protein crystallized at 20°C and diffraction data from the crystals were collected at a resolution of 1.9 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 116.8, b = 121.7, c = 318.0 Å. Analysis of the crystal structure revealed its conformation to be similar to the organization of the aldehyde dehydrogenase superfamily with three domains: the catalytic, NAD+-binding and bridging domains. The crystal structure of NahF from P. putida G7 was found to be the best structural homologue of SALDAP, even though the enzymes share only 48% amino-acid identity. Interestingly, a carboxylic acid (protocatechuic acid) was found to be a putative ligand of the enzyme and differential scanning fluorimetry was employed to confirm ligand binding. These findings open up the possibility of studying the mechanism(s) of product inhibition and biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.

Background Galactose epimerase (GALE) deficiency is a rare hereditary disorder of galactose metabolism with only a few cases described in the literature. This study aims to present the data of patients with GALE deficiency from different countries included through the Galactosemia Network to further expand the existing knowledge and review the current diagnostic strategy, treatment and follow-up of this not well characterized entity. Methods Observational study collecting medical data from December 2014 to April 2022 of 22 not previously reported patients from 14 centers in 9 countries. Patients were classified as generalized or non-generalized based on their genotype, enzyme activities in different tissues and/or clinical picture and professional judgment of the treating physician. Results In total 6 patients were classified as generalized and 16 as non-generalized. In the generalized group, acute neonatal illness was reported in 3, cognitive and developmental delays were present in 5 and hearing problems were reported in 3. Four generalized patients were homozygous for the genetic variant NM_001008216.2:c.280G > A (p.Val94Met). In the non-generalized group, no clearly related symptoms were found. Ten novel genetic variants were reported in this study population. Conclusion The phenotypic spectrum of GALE deficiency ranges from asymptomatic to severe. The generalized patients have a phenotype that is in line with the 9 described cases in the literature and prescribing dietary interventions is the cornerstone for treatment. In the non-generalized group, treatment advice is more difficult. To be able to offer proper counseling, in addition to red blood cell enzyme activity, genetic studies, transferrin glycoform analysis and enzymatic measurements in fibroblasts are recommended. Due to lack of facilities, additional enzymatic testing is not common practice in many centers nor a tailored long-term follow-up is performed.

This research was funded by the ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency (Grant RTI2018-096246-B-I00, to A.L.P., PID2019-110900GBI00 to M.M. and SAF2015-69796 to E.S.), Consejeriia de Economiia, Conocimiento, Empresas y Universidad, Junta de Andalucia (Grant P18-RT-2413, to A.L.P.), and the Government of AragonFEDER (Grant E35-20R to M.M.)., HIF-1 alpha is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1 alpha inhibition represents an interesting target for anti-cancer therapy. It was recently shown that the HIF-1 alpha interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1 alpha as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1 alpha are beginning to be unraveled, in this review we discuss: (1) Structure-function relationships of HIF-1 alpha; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1 alpha levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1 alpha, p53 and p73 alpha. This knowledge is then further discussed in the context of potentially targeting the intracellular stability of HIF-1 alpha by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals., ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00 PID2019-110900GBI00 SAF2015-69796, Junta de Andalucia P18-RT-2413, Government of AragonFEDER E35-20R

Biofuels have the capacity to contribute to carbon dioxide emission reduction and to energy security as oil reserves diminish and/or become concentrated in politically unstable regions. However, challenges exist in obtaining the maximum yield from industrial fermentations. One challenge arises from the nature of alcohols. These compounds are chaotropic (i.e. causes disorder in the system) which causes stress in the microbes producing the biofuel. Brewer’s yeast (Saccharomyces cerevisiae) typically cannot grow at ethanol concentration much above 17% (v/v). Mitigation of these properties has the potential to increase yield. Previously, we have explored the effects of chaotropes on model enzyme systems and attempted (largely unsuccessfully) to offset these effects by kosmotropes (compounds which increase the order of the system, i.e. the “opposite” of chaotropes). Here we present some theoretical results which suggest that high molecular mass polyethylene glycols may be the most effective kosmotropic additives in terms of both efficacy and cost. The assumptions and limitations of these calculations are also presented. A deeper understanding of the effects of chaotropes on biofuel-producing microbes is likely to inform improvements in bioethanol yields and enable more rational approaches to the “neutralisation” of chaotropicity.

Hemoglobinopathies are inherited diseases that impair the structure and function of the oxygen-carrying pigment hemoglobin (Hb). Adult Hb consists of two α and two β subunits. α-Thalassemia (α-thal) affects the genes that code for the α-globin chains, HBA1 and HBA2. Mutations can result in asymptomatic, mild or severe outcomes depending on several factors, such as mutation type, number of mutations and the location at which they occur. PredictSNP was used to estimate whether every possible single nucleotide polymorphism (SNP) would have a neutral or deleterious effect on the protein. These results were then used to create a plot of predicted tolerance to change for each residue in the protein. Tolerance to change was negatively correlated with the residue's sequence conservation score. The PredictSNP data were compared to clinical reports of 110 selected variants in the literature. There were 29 disagreements between the two data types. Some of these could be resolved by considering the role of the affected residue in binding other molecules. The three-dimensional structures of some of these variant proteins were modeled. These models helped explain variants which affect heme binding. We predict that where a point mutation alters a residue that is intolerant to change, is well conserved and or involved in interactions, it is likely to be associated with disease. Overall, the data from this study could be used alongside biochemical and clinical data to assess novel α-globin variants.

Galactosemia is the inherited inability to metabolise galactose. The most common results from a lack of galactose 1-phosphate uridylyltransferase activity. The current treatment, removal of galactose from the diet, is inadequate and often fails to prevent long-term complications. Since 2015, three patents have been filed describing novel therapies. These are: the use of aldose reductase inhibitors to reduce cataracts and, possibly, other symptoms; salubrinal to stimulate cellular stress responses; mRNA therapy to increase cellular galactose 1-phosphate uridylyltransferase activity. The viability of all three is supported by academic studies. The potential and drawbacks of all three are discussed and evaluated.

Introduction: Galactosemia results from mutations in genes which code for enzymes involved in the metabolism of galactose. It has a wide range of symptoms ranging from the relatively mild (early on...

Chaotropes are compounds which cause the disordering, unfolding and denaturation of biological macromolecules. It is the chaotropicity of fermentation products that often acts as the primary limiting factor in ethanol and butanol fermentations. Since ethanol is mildly chaotropic at low concentrations, it prevents the growth of the producing microbes via its impacts on a variety of macromolecular systems and their functions. Kosmotropes have the opposite effect to chaotropes and we hypothesised that it might be possible to use these to mitigate chaotrope-induced inhibition of Saccharomyces cerevisiae growth. We also postulated that kosmotrope-mediated mitigation of chaotropicity is not quantitatively predictable. The chaotropes ethanol and urea, and compatible solutes glycerol and betaine (kosmotrope), and the highly kosmotropic salt ammonium sulphate all inhibited the growth rate of Saccharomyces cerevisiae in the concentration range 5-15%. They resulted in increased lag times, decreased maximum specific growth rates, and decreased final optical densities. Surprisingly, neither the stress protectants nor ammonium sulphate reduced the inhibition of growth caused by ethanol. Whereas, in some cases, compatible solutes and kosmotropes mitigated against the inhibitory effects of urea. However, this effect was not mathematically additive from the quantification of chao-/kosmotropicity of each individual compound. The potential effects of glycerol, betaine and/or ammonium sulphate may have been reduced or masked by the metabolic production of compatible solutes. It may nevertheless be that the addition of kosmotropes to fermentations which produce chaotropic products can enhance metabolic activity, growth rate, and/or product formation.

Mevalonate Kinase (MVK) catalyses the ATP-Mg2+ mediated phosphate transfer of mevalonate to produce mevalonate 5-phosphate and is a key kinase in the mevalonate pathway in the biosynthesis of isopentenyl diphosphate, the precursor of isoprenoid-based biofuels. However, the crystal structure in complex with the native substrate mevalonate, ATP and Mg2+ has not been resolved, which has limited the understanding of its reaction mechanism and therefore its application in the production of isoprenoid-based biofuels. Here using molecular docking, molecular dynamic (MD) simulations and a hybrid QM/MM study, we revisited the location of Mg2+ resolved in the crystal structure of MVK and determined a catalytically competent MVK structure in complex with the native substrate mevalonate and ATP. We demonstrated that significant conformational change on a flexilble loop connecting the α6 and α7 helix is induced by the substrate binding. Further, we found that Asp204 is coordinated to the Mg2+ ion. Arg241 plays a crucial role in organizing the triphosphoryl tail of ATP for in-line phosphate transfer and stabilizing the negative charge that accumulates at the β,γ-bridging oxygen of ATP upon bond cleavage. Remarkably, we revealed that the phosphorylation of mevalonate catalyzed by MVK occurs via a direct phosphorylation mechanism, instead of the conventionally postulated catalytic base mechanism. The catalytically competent complex structure of MVK as well as the mechanism of reaction will pave the way for the rational engineering of MVK to exploit its applications in the production of biofuels.

Type IV galactosemia is a recently discovered inherited metabolic disease. It is caused by mutations in the GALM gene which result in reduced activity of the enzyme galactose mutarotase. This enzyme catalyses the interconversion of the α- and β-anomers of d-galactose and some other monosaccharides. Human galactose mutarotase is monomeric and its structure is largely composed of β-sheets. The catalytic mechanism requires a histidine residue acting as an acid, and a glutamate acting as a base. Together, these residues open the pyranose ring of d-galactose enabling free rotation of the bond between the first two carbon atoms in the monosaccharide. This can cause reversal of the configuration of the hydroxyl group attached to carbon 1. Type IV galactosemia manifests with similar symptoms to type II galactosemia (galactokinase deficiency), i.e. early onset cataracts. However, as a recently discovered disease, the longer-term consequences are unknown. The physiological role, if any, of galactose mutarotase's reactions with other monosaccharides are not yet known. The possible associations with other proteins also require further investigation.

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth / development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 hour exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

Chaotropicity has long been recognised as a property of some compounds. Chaotropes tend to disrupt non-covalent interactions in biological macromolecules (e.g. proteins and nucleic acids) and supramolecular assemblies (e.g. phospholipid membranes). This results in the destabilisation and unfolding of these macromolecules and assemblies. Unsurprisingly, these compounds are typically harmful to living cells since they act against multiple targets, comprising cellular integrity and function. Kosmotropes are the opposite of chaotropes and these compounds promote the ordering and rigidification of biological macromolecules and assemblies. Since many biological macromolecules have optimum levels of flexibility, kosmotropes can also inhibit their activity and can be harmful to cells. Some products of industrial fermentations, most notably alcohols, are chaotropic. This property can be a limiting factor on rates of production and yields. It has been hypothesised that the addition of kosmotropes may mitigate the chaotropicity of some fermentation products. Some microbes naturally adapt to chaotropic environments by producing kosmotropic compatible solutes. Exploitation of this in industrial fermentations has been hampered by scientific and economic issues. The cost of the kosmotropes and their removal during purification needs to be considered. We lack a complete understanding of the chemistry of chaotropicity and a robust, quantitative framework for estimating overall chaotropicities of mixtures. This makes it difficult to predict the amount of kosmotrope required to neutralise the chaotropicity. This review considers examples of industrial fermentations where chaotropicity is an issue and suggests possible mitigations.

Praziquantel is a remarkably effective drug for the treatment of schistosomiasis. It has few side effects, some of which have been attributed to its inactive enantiomer. Few, if any, verified cases of drug resistance have been reported in a clinical setting. The preponderance of scientific evidence suggests that the drug works by dysregulating calcium homeostasis in the worm. Voltage-gated calcium channels have been proposed as the main pharmacological target of praziquantel, although no direct evidence of interaction with this protein is available. Here, the biochemical pharmacology of praziquantel is briefly reviewed and a hypothesis for its mechanism proposed. This hypothesis suggests that the drug works, in part, by disrupting an interaction between a voltage-gated calcium channel (SmCav1B) and an accessory protein, SmTAL1.

Tay‒Sachs disease (TSD), a deficiency of β-hexosaminidase A (Hex A), is a rare but debilitating hereditary metabolic disorder. Symptoms include extensive neurodegeneration and often result in death in infancy. We report an in silico study of 42 Hex A variants associated with the disease. Variants were separated into three groups according to the age of onset: infantile (n=28), juvenile (n=9) and adult (n=5). Protein stability, aggregation potential and the degree of conservation of residues were predicted using a range of in silico tools. We explored the relationship between these properties and the age of onset of TSD. There was no significant relationship between protein stability and disease severity or between protein aggregation and disease severity. Infantile TSD had a significantly higher mean conservation score than nondisease associated variants. This was not seen in either juvenile or adult TSD. This study has established that the degree of residue conservation may be predictive of infantile TSD. It is possible that these more highly conserved residues are involved in trafficking of the protein to the lysosome. In addition, we developed and validated software tools to automate the process of in silico analysis of proteins involved in inherited metabolic diseases. Further work is required to identify the function of well-conserved residues to establish an in silico predictive model of TSD severity.

Biofuels have the capacity to contribute to carbon dioxide emission reduction and to energy security as oil reserves diminish and/or become concentrated in politically unstable regions. However, challenges exist in obtaining the maximum yield from industrial fermentations. One challenge arises from the nature of alcohols. These compounds are chaotropic (i.e. causes disorder in the system) which causes stress in the microbes producing the biofuel. Brewer's yeast (Saccharomyces cerevisiae) typically cannot grow at ethanol concentration much above 17% (v/v). Mitigation of these properties has the potential to increase yield. Previously, we have explored the effects of chaotropes on model enzyme systems and attempted (largely unsuccessfully) to offset these effects by kosmotropes (compounds which increase the order of the system, i.e. the "opposite" of chaotropes). Here we present some theoretical results which suggest that high molecular mass polyethylene glycols may be the most effective kosmotropic additives in terms of both efficacy and cost. The assumptions and limitations of these calculations are also presented. A deeper understanding of the effects of chaotropes on biofuel-producing microbes is likely to inform improvements in bioethanol yields and enable more rational approaches to the "neutralisation" of chaotropicity.

HIF-1α is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1α inhibition represents an interesting target for anti-cancer therapy. It was recently shown that HIF-1α interaction with NQO1 inhibits its proteasomal degradation, thus suggesting that targeting the stability of NQO1 could led to destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, we review here our current knowledge on the intracellular functions and stability of NQO1, its pharmacological modulation by small ligands, and the molecular determinants of its roles as a chaperone of many different proteins including cancer-associated factors such as p53 and p73α. This knowledge is then discussed in the context of potentially targeting the intracellular stability of HIF-1α by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies.

Galactosemia is an inherited metabolic disease affecting enzymes of the Leloir pathway of galactose metabolism. There are four types: type I (galactose 1-phosphate uridylyltransferase; GALT), type II (galactokinase; GALK1), type III (uridine diphosphate [UDP]-galactose 4’-epimerase; GALE), and type IV (galactose mutarotase; GALM). The range of symptoms is wide, ranging from almost asymptomatic to life-threatening damage to the liver, kidneys, and brain. Severely affected patients suffer cognitive disabilities. Current treatment relies on the restriction of galactose (and its precursors such as lactose) from the diet. This is imperfect and often slows, rather than prevents, the development of symptoms. Potential new treatment approaches include enzyme replacement therapy (ERT), inhibition of galactokinase, antioxidants, phosphate supplementation, and pharmacological chaperones. ERT has been successfully applied in other diseases, but it is likely that the proteins would need to be delivered to the brain to be fully effective. This would present problems with currently available technologies. Antioxidant therapy and phosphate supplementation would treat downstream effects of the disease and not the fundamental causes. They may be useful in conjunction with galactose restriction but are unlikely to provide a complete solution for the most severely affected patients. Galactokinase inhibition has the most underpinning research with several promising compounds identified. It has yet to be tested in clinical trials. Pharmacological chaperone therapy offers the opportunity to correct protein folding and restore activity. Challenges include development of suitable assays, potential for long-term toxicity of drugs that may be taken for a lifetime, and identifying compounds that work with different disease-associated variants.

Praziquantel is a remarkably effective drug for the treatment of schistosomiasis. It has few side effects, some of which have been attributed to its inactive enantiomer. Few, if any, verified cases of drug resistance have been reported in a clinical setting. The preponderance of scientific evidence suggests that the drug works by dysregulating calcium homeostasis in the worm. Voltage-gated calcium channels have been proposed as the main pharmacological target of praziquantel, although no direct evidence of interaction with this protein is available. Here, the biochemical pharmacology of praziquantel is briefly reviewed and a hypothesis for its mechanism proposed. This hypothesis suggests that the drug works, in part, by disrupting an interaction between a voltage-gated calcium channel (SmCav1B) and an accessory protein, SmTAL1.

Background: Saccharomyces cerevisiae var. boulardii is the only yeast species with probiotic properties. It is considered to have therapeutic significance in gastrointestinal disorders. In the present study, a comparative physiological study between this yeast and Saccharomyces cerevisiae (BY4742) was performed by evaluating two prominent traits of probiotic species, responses to different stress conditions and antioxidant capacity. A global metabolite profile was also developed aiming to identify which therapeutically important secondary metabolites are produced.; Results: Saccharomyces cerevisiae var. boulardii showed no significant difference in growth patterns but greater stress tolerance compared to S. cerevisiae. It also demonstrated a six- to 10-fold greater antioxidant potential (judged by the 1,1-diphenyl-2-picrylhydrazyl assay), with a 70-fold higher total phenolic content and a 20-fold higher total flavonoid content in the extracellular fraction. These features were clearly differentiated by principal component analysis and further indicated by metabolite profiling. The extracellular fraction of the S. cerevisiae var. boulardii cultures was found to be rich in polyphenolic metabolites: vanillic acid, cinnamic acid, phenyl ethyl alcohol (rose oil), erythromycin, amphetamine and vitamin B6 , which results in the antioxidant capacity of this strain.; Conclusion: The present study presents a new perspective for differentiating the two genetically related strains of yeast, S. cerevisiae and S. cerevisiae var. boulardii by assessing their metabolome fingerprints. In addition to the correlation of the phenotypic properties with the secretory metabolites of these two yeasts, the present study also emphasizes the potential to exploit S. cerevisiae var. boulardii in the industrial production of these metabolites. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme’s ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound’s well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

Galactokinase catalyses the ATP-dependent phosphorylation of galactose and structurally related sugars. The enzyme has attracted interest as a potential biocatalyst for the production of sugar 1-phosphates and several attempts have been made to broaden its specificity. In general, bacterial galactokinases have wider substrate ranges than mammalian ones. The enzymes from Escherichia coli and Lactococcus lactis have received particular attention and a number of variants with increased promiscuity have been identified. Here, we present a molecular dynamics study designed to investigate the molecular causes of the wider substrate ranges of these enzymes and their variants with particular reference to protein mobility. Some regions close to the active site of the enzyme have different structures in the bacterial enzymes compared to the human one. Alterations known to increase the substrate range (e.g. Y371H in the E. coli enzyme), tend to alter the conformation of a key α-helical region (residues 216–232 in the E. coli enzyme). The equivalent helix in the human enzyme has previously been predicted to be altered in variants which affect catalytic activity or protein stability. This helix appears to be a key region in galactokinases from a range of species and may represent an interesting target for future attempts to broaden the specificity of galactokinases.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a multi-functional protein that catalyses the reduction of quinones (and other molecules), thus playing roles in xenobiotic detoxification and redox balance, and also has roles in stabilising apoptosis regulators such as p53. The structure and enzymology of NQO1 is well-characterised, showing a substituted enzyme mechanism in which NAD(P)H binds first and reduces an FAD cofactor in the active site, assisted by a charge relay system involving Tyr-155 and His-161. Protein dynamics play important role in physio-pathological aspects of this protein. NQO1 is a good target to treat cancer due to its overexpression in cancer cells. A polymorphic form of NQO1 (p.P187S) is associated with increased cancer risk and certain neurological disorders (such as multiple sclerosis and Alzheimer´s disease), possibly due to its roles in the antioxidant defence. p.P187S has greatly reduced FAD affinity and stability, due to destabilization of the flavin binding site and the C-terminal domain, which leading to reduced activity and enhanced degradation. Suppressor mutations partially restore the activity of p.P187S by local stabilization of these regions, and showing long-range allosteric communication within the protein. Consequently, the correction of NQO1 misfolding by pharmacological chaperones is a viable strategy, which may be useful to treat cancer and some neurological conditions, targeting structural spots linked to specific disease-mechanisms. Thus, NQO1 emerges as a good model to investigate loss of function mechanisms in genetic diseases as well as to improve strategies to discriminate between neutral and pathogenic variants in genome-wide sequencing studies.

A recent paper in Bioscience Reports (BSR20182189) describes the discovery of an interaction between the motor protein myosin Va and the metabolic enzyme spermine synthase. Myosin Va is a molecular motor which plays a key role in vesicle transport. Mutations in the gene which encodes this protein are associated with Griscelli syndrome type 1 and the ‘dilute’ phenotype in animals. Spermine synthase catalyzes the conversion of spermidine to spermine. This largely cytoplasmic enzyme can also be localized to the soluble fraction in exosomes. Mutations in the spermine synthase gene are associated with Snyder Robinson mental retardation syndrome. The interaction between the two proteins was detected using the yeast two hybrid method and verified by microscale thermophoresis of recombinant proteins. Knockdown of the MYO5A gene reduced the expression of mRNA coding for spermine synthase. The amount of this transcript was also reduced in cells derived from a patient with Griscelli syndrome type 1. This suggests that, in addition to a direct physical interaction between the two proteins, myosin Va also modulates the transcription of the spermine synthase gene. The mechanism for this modulation is currently unknown. These findings have implications for Griscelli syndrome type 1 and Snyder Robinson mental retardation syndrome. They also suggest that interactions between myosin Va and soluble exosome proteins such as spermine synthase may be important in the mechanism of exosome transport.

Parasitic diseases caused by helminths (worms) represent a major burden on humanity with hundreds of millions of people infected worldwide. However, there are relatively few drugs to treat these diseases, and resistance is emerging to some of these. Therefore, there is a pressing need to characterize proteins from helminths as potential drug targets. Calcium signalling proteins represent attractive targets due to the vital nature of properly regulated calcium-mediated signalling and the presence of unusual calcium-binding proteins in helminths. Here we present methods to characterize these proteins in terms of their ion-binding properties, drug-binding properties, and oligomeric state, including a method to correct for the effects of non-spherical proteins in analytical gel filtration. In addition we present an overview of their recombinant expression and purification and methods to predict their structures.

Parasitic diseases caused by helminths (worms) represent a major burden on humanity with hundreds of millions of people infected worldwide. However, there are relatively few drugs to treat these diseases, and resistance is emerging to some of these. Therefore, there is a pressing need to characterize proteins from helminths as potential drug targets. Calcium signalling proteins represent attractive targets due to the vital nature of properly regulated calcium-mediated signalling and the presence of unusual calcium-binding proteins in helminths. Here we present methods to characterize these proteins in terms of their ion-binding properties, drug-binding properties, and oligomeric state, including a method to correct for the effects of non-spherical proteins in analytical gel filtration. In addition we present an overview of their recombinant expression and purification and methods to predict their structures.

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-6 (pp-GalNAc-T6) is a member of the N-acetyl-D-galactosamine transferase family. It catalyzes the addition of N-acetyl-D-galactosamine to proteins, often the first step in O-glycosylation of proteins. Glycosylated proteins play important roles in vivo in the cell membrane. These are often involved in cell-cell adhesion, cytoskeleton regulation and immune recognition. pp-GalNAc-T6 has been shown to be upregulated in a number of types of cancer. Abnormally glycosylated forms of mucin 1 (a substrate of the enzyme), are used clinically as a biomarker for breast cancer. There is potential for other products of the pp-GalNAc- T6 catalyzed reaction to be used. It is also possible that pp-GalNAc-T6 itself could be used as a biomarker, since levels of this protein tend to be low in non-malignant tissues. pp- GalNAc-T6 has been implicated in malignant transformation and metastasis of cancer cells. As such, it has considerable potential as a target for chemotherapy. To date, no selective inhibitors of the enzyme have been identified. However, general inhibitors of the enzyme family result in reduced cell surface O-linked glycosylation and induce apoptosis in cultured cells. Thus, a selective inhibitor of pp-GalNAc-T6 is likely to target cancer cells and could be developed into a novel anticancer therapy.

IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an antioxidant and detoxifying enzyme involved in the two-electron reduction of a wide variety of quinones. As a non-enzymatic function, it is involved in the stabilization of several tumour suppressors such as p53, p33 and p73α. NQO1 is overexpressed in several types of tumours, and two common polymorphisms are associated with increased cancer risk, making NQO1 a potential target for new cancer treatments. Here we review the structural and enzymological properties of NQO1, as well as its roles in cancer development and treatment. Particularly, we focus on recent developments on the understanding of the molecular basis leading to loss-of-function in cancer-associated polymorphisms, and propose new approaches to target these molecular defects to develop new pharmacological agents to rescue them. We will focus on pharmacological therapies aimed at correcting the abnormal properties of polymorphic proteins (such as protein stability and dynamics) and modulating intracellular factors leading to loss-of-function (such as accelerated proteasomal degradation).

Proteins are highly mobile structures. In addition to gross conformational changes occurring on, for example, ligand binding, they are also subject to constant thermal motion. The mobility of a protein varies through its structure and can be modulated by ligand binding and other events. It is becoming increasingly clear that this mobility plays an important role in key functions of proteins including catalysis, allostery, cooperativity, and regulation. Thus, in addition to an optimum structure, proteins most likely also require an optimal dynamic state. Alteration of this dynamic state through protein engineering will affect protein function. A dramatic example of this is seen in some inherited metabolic diseases where alternation of residues distant from the active site affects the mobility of the protein and impairs function. We postulate that using molecular dynamics simulations, experimental data or a combination of the two, it should be possible to engineer the mobility of active sites. This may be useful in, for example, increasing the promiscuity of enzymes. Thus, a paradigm for protein engineering is suggested in which the mobility of the active site is rationally modified. This might be combined with more "traditional" approaches such as altering functional groups in the active site.

A coding sequence (CD36-03230) from the yeast Candida dubliniensis had been previously annotated as a vanillin dehydrogenase (VDH). The corresponding protein (CD36-03230p) was recombinantly expressed in Escherichia coli and analysed. The protein is most likely a tetramer in solution as judged by crosslinking and gel filtration experiments. CD36-03230p is an active aldehyde dehydrogenase favouring cyclic and aromatic substrates. Positive cooperativity and substrate inhibition were observed with some substrates. The redox cofactor NADP+ and substrates affected the thermal stability of the protein. Interestingly, the enzyme had no detectable activity with vanillin suggesting that the annotation is incorrect. It has been previously hypothesized that a methionine residue at a key position in the active site of yeast aldehyde dehydrogenases sterically hinders cyclic substrates and restricts specificity to aliphatic aldehydes. Molecular modeling of CD36-03230p demonstrates that it has an isoleucine residue (Ile-156) at this position, further strengthening this hypothesis.

Fosfomycin Resistance Kinase A (FomA) catalyzes the phosphorylation of fosfomycin, which is an effective antibiotic for treating urinary tract infections. Understanding the chemical reaction mechanism is essential for developing strategies to counter the resistance of fosfomycin in clinical settings. Here the catalytic mechanism of FomA was investigated using molecular dynamic simulations in conjunction with quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Our QM/MM study disclosed that the phosphorylation reaction catalyzed by FomA follows a dissociative mechanism, in contrast to the previously proposed associative mechanism. In addition, we found that His58, a characteristic residue in the AAK family, plays a key role in positioning the phosphate group of fosfomycin in the transition state. Molecular dynamic simulations revealed the important roles of Lys9 and Lys18 in arranging the nucleotide for phosphate transfer. Furthermore, we identified a four-membered water network mediated by Asp171 and Ser13 that is critical in ordering ATP for phosphate transfer. The active structure and reaction mechanism of FomA will provide valuable insights for developing new strategies to tackle the resistance to Fosfomycin-based antibiotic therapies.

The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins - SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCav1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels.

Mevalonate kinase (MVK) catalyses the phosphorylation of mevalonate. Deficiency of MVK is associated with two rare periodic fever syndromes, mevalonic aciduria (MA), a severe form and hyper-immunoglobulin-D syndrome (HIDS), a milder form. An in silico approach was used to analyse the physicochemical and structural effects of 47 disease-associated variants of MVK. A further 20 variants, which are present in human genome databases, were also analysed. Variants associated with MA are clustered into a "hotspot" consisting of residues 8-35 and 234-338 and tended to result in a prediction of severely reduced protein stability. Four of the uncharacterised variants, p.H24P, p.G198R, p. R253W, and p.G335S, were likely to be associated with MA. This method could be used as the basis for initial predictions of severity when new MVK variants are discovered.

SUMMARYFhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.

Fermentation products can chaotropically disorder macromolecular systems and induce oxidative stress, thus inhibiting biofuel production. Recently, the chaotropic activities of ethanol, butanol and vanillin have been quantified (5.93, 37.4, 174kJ kg(-1)m(-1) respectively). Use of low temperatures and/or stabilizing (kosmotropic) substances, and other approaches, can reduce, neutralize or circumvent product-chaotropicity. However, there may be limits to the alcohol concentrations that cells can tolerate; e.g. for ethanol tolerance in the most robust Saccharomyces cerevisiae strains, these are close to both the solubility limit (

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine: glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases., Spanish Ministry of Economy and Competitiveness, MINECO (BIO 2015 66426-R to JMSR, CTQ 2015-64445-R to JLN, ‘Factoría Española de Cristalización’, Consolider-Ingenio 2010 to JAG and SAF2015-69796 to ES), Junta de Andalucia (P11-CTS-07187 to ALP) and FEDER fund

Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met177, compared with the equivalent residue Leu196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity.

Mutations causing single amino acid exchanges can dramatically affect protein stability and function, leading to disease. In this chapter, we will focus on several representative cases in which such mutations affect protein stability and function leading to cancer. Mutations in BRAF and p53 have been extensively characterized as paradigms of loss-of-function/gain-of-function mechanisms found in a remarkably large fraction of tumours. Loss of RB1 is strongly associated with cancer progression, although the molecular mechanisms by which missense mutations affect protein function and stability are not well known. Polymorphisms in NQO1 represent a remarkable example of the relationships between intracellular destabilization and inactivation due to dynamic alterations in protein ensembles leading to loss of function. We will review the function of these proteins and their dysfunction in cancer and then describe in some detail the effects of the most relevant cancer-associated single amino exchanges using a translational perspective, from the viewpoints of molecular genetics and pathology, protein biochemistry and biophysics, structural, and cell biology. This will allow us to introduce several representative examples of natural and synthetic small molecules applied and developed to overcome functional, stability, and regulatory alterations due to cancer-associated amino acid exchanges, which hold the promise for using them as potential pharmacological cancer therapies.

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of

NAD(P)H quinone oxidoreductase 1 is involved in antioxidant defence and protection from cancer, stabilizing the apoptosis regulator p53 towards degradation. Here, we studied the enzymological, biochemical and biophysical properties of two cancer-associated variants (p.R139W and p.P187S). Both variants (especially p.187S) have lower thermal stability and greater susceptibility to proteolysis compared to the wild-type. p.P187S also has reduced activity due to a lower binding affinity for the FAD cofactor as assessed by activity measurements and direct titrations. Native gel electrophoresis and dynamic light scattering also suggest that p.P187S has a higher tendency to populate unfolded states under native conditions. Detailed thermal stability studies showed that all variants irreversibly denature causing dimer dissociation, while addition of FAD restores the stability of the polymorphic forms to wild-type levels. The kinetic destabilization induced by polymorphisms as well as the kinetic protection exerted by FAD was confirmed by measuring denaturation kinetics at temperatures close to physiological. Our data suggest that the main molecular mechanisms associated with these cancer-related variants are their low binding affinity for FAD and/or kinetic instability. Thus, pharmacological chaperones may be useful in the treatment of patients bearing these polymorphisms.

Since a key requirement of known life forms is available water (water activity; aw), recent searches for signatures of past life in terrestrial and extraterrestrial environments have targeted places known to have contained significant quantities of biologically available water. However, early life on Earth inhabited high-salt environments, suggesting an ability to withstand low water-activity. The lower limit of water activity that enables cell division appears to be ∼ 0.605 which, until now, was only known to be exhibited by a single eukaryote, the sugar-tolerant, fungal xerophile Xeromyces bisporus. The first forms of life on Earth were, though, prokaryotic. Recent evidence now indicates that some halophilic Archaea and Bacteria have water-activity limits more or less equal to those of X. bisporus. We discuss water activity in relation to the limits of Earth's present-day biosphere; the possibility of microbial multiplication by utilizing water from thin, aqueous films or non-liquid sources; whether prokaryotes were the first organisms able to multiply close to the 0.605-aw limit; and whether extraterrestrial aqueous milieux of ≥ 0.605 aw can resemble fertile microbial habitats found on Earth.

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson&rsquo, s and Alzheimer&acute, s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0 °C). The steady-state kinetic parameters are high (Km for dihydroxyacetone phosphate is 0.51 mM; Km for glyceraldehyde 3-phosphate is 1.1 mM; kcat for dihydroxyacetone phosphate is 7800 s(-1) and kcat for glyceraldehyde 3-phosphate is 6.9s(-1)).

UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr-157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosemine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.

GHMP kinases are a group of structurally related small molecule kinases. They have been found in all kingdoms of life and are mostly responsible for catalyzing the ATP-dependent phosphorylation of intermediary metabolites. Although the GHMP kinases are of clinical, pharmaceutical, and biotechnological importance, the mechanism of GHMP kinases is controversial. A catalytic base mechanism was suggested for mevalonate kinase that has a structural feature of the γ-phosphate of ATP close to an aspartate residue; however, for one GHMP family member, homoserine kinase, where the residue acting as general base is absent, a direct phosphorylation mechanism was suggested. Furthermore, it was proposed by some authors that all the GHMP kinases function by a similar mechanism. This controversy in mechanism has limited our ability to exploit these enzymes as drug targets and in biotechnology. Here the phosphorylation reaction mechanism of the human galactokinase, a member of the GHMP kinase family, was investigated using molecular dynamics simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B3LYP-D/AMBER99). The reaction coordinates were localized by potential energy scan using an adiabatic mapping method. Our results indicate that a highly conserved Glu174 captures Arg105 in the proximity of the α-phosphate of ATP, forming a H-bond network; therefore, the mobility of ATP in the large oxyanion hole is restricted. Arg228 functions to stabilize the negative charge developed at the β,γ-bridging oxygen of the ATP during bond cleavage. The reaction occurs via a direct phosphorylation mechanism, and the Asp186 in the proximity of ATP does not directly participate in the reaction pathway. Because Arg228 is not conserved among GHMP kinases, reagents which form interactions with Arg228, and therefore can interrupt its function in phosphorylation, may be developed into potential selective inhibitors for galactokinase.