Galactokinase deficiency: lessons from the GalNet registry.

PURPOSE: Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype. METHODS: Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020. RESULTS: Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial. CONCLUSION: The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.

In Silico Analysis of the Effects of Point Mutations on α-Globin: Implications for α-Thalassemia.

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 ß 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.

The roles and applications of chaotropes and kosmotropes in industrial fermentation processes.

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.

Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution.

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.

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1).

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

Catalytic mechanism of mevalonate kinase revisited, a QM/MM study.

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 dynamics (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 flexible 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 ß,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.

Evidence for chaotropicity/kosmotropicity offset in a yeast growth model.

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.

LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/ß-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/ß-catenin signaling or cell proliferation in vivo Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell-cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell-cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell-cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1-Rac1 pathway to strengthen cell-cell adhesion in normal adult crypt stem cells and colon cancer cells.

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications.

Galactokinase catalyses the site- and stereospecific phosphorylation of α-d-galactose. As such it has attracted interest as a biocatalyst for the introduction of phosphate groups into monosaccharides. However, attempts to broaden the substrate range of human galactokinase have generally resulted in substantially reduced activity. The enzyme also has biotechnological potential in enzyme replacement therapy (ERT) for type II galactosaemia. The return-to-consensus approach can be used to identify residues that can be altered to increase protein stability and enzyme activity. This approach identified six residues of potential interest in human galactokinase. Some of the single consensus variants (M60V, D268E, A334S and G373S) increased the catalytic turnover of the enzyme, but none resulted in improved stability. When all six changes were introduced into the protein (M60V/M180V/D268E/A334S/R366Q/G373S), thermal stability was increased. Molecular dynamics simulations suggested that these changes altered the protein's conformation at key sites. The number of salt bridges and hydrogen bonds was also increased. Combining the six consensus variations with Y379W (a variant with greater substrate promiscuity) increased the stability of this variant and its turnover towards some substrates. Thus, the six consensus variants can be used to stabilise catalytically interesting variants of human galactokinase and might also be useful if the protein were to be used in ERT.

Water-mediated network in the resistance mechanism of fosfomycin.

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.

Modulation of the mobility of a key region in human galactokinase: Impacts on catalysis and stability.

Galactokinase catalyses the phosphorylation of α-d-galactose and some structurally related monosaccharides. The enzyme is of interest due to its potential as a biocatalyst for the production of sugar 1-phosphates and due to its involvement in the inherited metabolic disease type II galactosemia. It has been previously shown that a region (residues 231-245) in human galactokinase often has altered mobility when active site residues are varied. We hypothesised that the reverse may be true and that designing changes to this region might affect the functioning of the active site of the enzyme. Focussing on four residues (Leu-231, Gln-242, Glu-244 and Glu-245) we conducted molecular dynamics simulations to explore the effects of changing these residues to glycine or serine. In most cases the variations resulted in local changes to the 231-245 region and global changes to the root mean squared fluctuation (RMSF) of the protein. The four serine variants were expressed as recombinant proteins. All had altered steady state enzyme kinetic parameters with α-d-galactose as a substrate. However, these changes were generally less than ten-fold in magnitude. Changes were also observed with 2-deoxy-α-d-galactose, α-d-galactosamine and α-d-talose as substrates, including (in some cases) loss of detectable activity, suggesting that these variations can tune the specificity of the enzyme. This study demonstrates that activity and specificity of human galactokinase can be modulated by variations designed to affect active site flexibility. It is likely that this principle can be generalised to other enzymes.

A galactokinase-like protein from the liver fluke Fasciola hepatica.

Galactokinase catalyses the ATP-dependent phosphorylation of galactose. A galactokinase-like sequence was identified in a Fasciola hepatica EST library. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein of approximately 50 kDa. The protein is monomeric, like galactokinases from higher animals, yeasts and some bacteria. The protein has no detectable enzymatic activity with galactose or N-acetylgalactosamine as a substrate. However, it does bind to ATP. Molecular modelling predicted that the protein adopts a similar fold to galactokinase and other GHMP kinases. However, a key loop in the active site was identified which may influence the lack of activity. Sequence analysis strongly suggested that this protein (and other proteins annotated as "galactokinase" in the trematodes Schistosoma mansoni and Clonorchis sinensis) are closer to N-acetylgalactosamine kinases. No other galactokinase-like sequences appear to be present in the genomes of these three species. This raises the intriguing possibility that these (and possibly other) trematodes are unable to catabolise galactose through the Leloir pathway due to the lack of a functional galactokinase.

Natural (and Unnatural) Small Molecules as Pharmacological Chaperones and Inhibitors in Cancer.

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.

IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES.

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.

Glycerol enhances fungal germination at the water-activity limit for life.

For the most-extreme fungal xerophiles, metabolic activity and cell division typically halts between 0.700 and 0.640 water activity (approximately 70.0-64.0% relative humidity). Here, we investigate whether glycerol can enhance xerophile germination under acute water-activity regimes, using an experimental system which represents the biophysical limit of Earth's biosphere. Spores from a variety of species, including Aspergillus penicillioides, Eurotium halophilicum, Xerochrysium xerophilum (formerly Chrysosporium xerophilum) and Xeromyces bisporus, were produced by cultures growing on media supplemented with glycerol (and contained up to 189 mg glycerol g dry spores-1 ). The ability of these spores to germinate, and the kinetics of germination, were then determined on a range of media designed to recreate stresses experienced in microbial habitats or anthropogenic systems (with water-activities from 0.765 to 0.575). For A. penicillioides, Eurotium amstelodami, E. halophilicum, X. xerophilum and X. bisporus, germination occurred at lower water-activities than previously recorded (0.640, 0.685, 0.651, 0.664 and 0.637 respectively). In addition, the kinetics of germination at low water-activities were substantially faster than those reported previously. Extrapolations indicated theoretical water-activity minima below these values; as low as 0.570 for A. penicillioides and X. bisporus. Glycerol is present at high concentrations (up to molar levels) in many types of microbial habitat. We discuss the likely role of glycerol in expanding the water-activity limit for microbial cell function in relation to temporal constraints and location of the microbial cell or habitat. The findings reported here have also critical implications for understanding the extremes of Earth's biosphere; for understanding the potency of disease-causing microorganisms; and in biotechnologies that operate at the limits of microbial function.

Characterisation of eppin function: expression and activity in the lung.

Eppin is a serine protease inhibitor expressed in male reproductive tissues.The aim of this study was to investigate the localisation and regulation of eppin expression in myeloid and epithelial cell lines, and explore its potential role as a multifunctional host defence protein.Using immunohistochemistry and Western blotting, eppin was detected in the lungs of patients with acute respiratory distress syndrome and cystic fibrosis lung disease. Expression of eppin in monocytic cells was unaffected by stimulation with Toll-like receptor agonists, cytokines and hormone receptor agonists. However, upregulated expression and secretion of eppin was observed following treatment of monocytes with epidermal growth factor. Incubation of recombinant eppin with monocytic cells resulted in significant inhibition of lipopolysaccharide-induced chemokine production. Furthermore, eppin inhibited lipopolysaccharide-induced NF-κB activation by a mechanism which involved accumulation of phosphorylated IκBα. In an in vivo model of lung inflammation induced by lipopolysaccharide, eppin administration resulted in decreased recruitment of neutrophils to the lung with a concomitant reduction in the levels of the neutrophil chemokine macrophage inflammatory protein-2.Overall, these results suggest a role for eppin outside of the reproductive tract and that eppin may have a role in the innate immune response in the lung.

Insight into the mechanism of galactokinase: Role of a critical glutamate residue and helix/coil transitions.

Galactokinase, the enzyme which catalyses the first committed step in the Leloir pathway, has attracted interest due to its potential as a biocatalyst and as a possible drug target in the treatment of type I galactosemia. The mechanism of the enzyme is not fully elucidated. Molecular dynamics (MD) simulations of galactokinase with the active site residues Arg-37 and Asp-186 altered predicted that two regions (residues 174-179 and 231-240) had different dynamics as a consequence. Interestingly, the same two regions were also affected by alterations in Arg-105, Glu-174 and Arg-228. These three residues were identified as important in catalysis in previous computational studies on human galactokinase. Alteration of Arg-105 to methionine resulted in a modest reduction in activity with little change in stability. When Arg-228 was changed to methionine, the enzyme's interaction with both ATP and galactose was affected. This variant was significantly less stable than the wild-type protein. Changing Glu-174 to glutamine (but not to aspartate) resulted in no detectable activity and a less stable enzyme. Overall, these combined in silico and in vitro studies demonstrate the importance of a negative charge at position 174 and highlight the critical role of the dynamics in to key regions of the protein. We postulate that these regions may be critical for mediating the enzyme's structure and function.

Antioxidant properties and global metabolite screening of the probiotic yeast Saccharomyces cerevisiae var. boulardii.

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.

A mysterious family of calcium-binding proteins from parasitic worms.

There is a family of proteins from parasitic worms which combine N-terminal EF-hand domains with C-terminal dynein light chain-like domains. Data are accumulating on the biochemistry and cell biology of these proteins. However, little is known about their functions in vivo Schistosoma mansoni expresses 13 family members (SmTAL1-SmTAL13). Three of these (SmTAL1, SmTAL2 and SmTAL3) have been subjected to biochemical analysis which demonstrated that they have different molecular properties. Although their overall folds are predicted to be similar, small changes in the EF-hand domains result in differences in their ion binding properties. Whereas SmTAL1 and SmTAL2 are able to bind calcium (and some other) ions, SmTAL3 appears to be unable to bind any divalent cations. Similar biochemical diversity has been seen in the CaBP proteins from Fasciola hepatica Four family members are known (FhCaBP1-4). All of these bind to calcium ions. However, FhCaBP4 dimerizes in the presence of calcium ions, FhCaBP3 dimerizes in the absence of calcium ions and FhCaBP2 dimerizes regardless of the prevailing calcium ion concentration. In both the SmTAL and FhCaBP families, the proteins also differ in their ability to bind calmodulin antagonists and related drugs. Interestingly, SmTAL1 interacts with praziquantel (the drug of choice for treating schistosomiasis). The pharmacological significance (if any) of this finding is unknown.

Galactokinase promiscuity: a question of flexibility?

Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering.