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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.

Human NQO1 [NAD(H):quinone oxidoreductase 1] is a multi-functional and stress-inducible dimeric protein involved in the antioxidant defense, the activation of cancer prodrugs and the stabilization of oncosuppressors. Despite its roles in human diseases, such as cancer and neurological disorders, a detailed characterization of its enzymatic cycle is still lacking. In this work, we provide a comprehensive analysis of the NQO1 catalytic cycle using rapid mixing techniques, including multiwavelength and spectral deconvolution studies, kinetic modeling and temperature-dependent kinetic isotope e ects (KIEs). Our results systematically support the existence of two pathways for hydride transfer throughout the NQO1 catalytic cycle, likely reflecting that the two active sites in the dimer catalyze two-electron reduction with di erent rates, consistent with the cooperative binding of inhibitors such as dicoumarol. This negative cooperativity in NQO1 redox activity represents a sort of half-of-sites activity. Analysis of KIEs and their temperature dependence also show significantly di erent contributions from quantum tunneling, structural dynamics and reorganizations to catalysis at the two active sites. Our work will improve our understanding of the e ects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment., ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00, Spanish Ministry of Science and Innovation-State Research Agency PID2019-103901GB-I00, Junta de Andalucía P11-CTS-07187 P18-RT-2413, Gobierno de Aragón-FEDER E35_20R

SmTAL1 is a calcium binding protein from the parasitic worm, Schistosoma mansoni. Structurally it is comprised of two domains - an N-terminal EF-hand domain and a C-terminal dynein light chain (DLC)-like domain. The protein has previously been shown to interact with the anti-schistosomal drug, praziquantel (PZQ). Here, we demonstrated that both EF-hands in the N-terminal domain are functional calcium ion binding sites. The second EF-hand appears to be more important in dictating affinity and mediating the conformational changes which occur on calcium ion binding. There is positive cooperativity between the four calcium ion binding sites in the dimeric form of SmTAL1. Both the EF-hand domain and the DLC-domain dimerise independently suggesting that both play a role in forming the SmTAL1 dimer. SmTAL1 binds non-cooperatively to PZQ and cooperatively to an IQ-motif from SmCav1B, a voltage-gated calcium channel. PZQ tends to strengthen this interaction, although the relationship is complex. These data suggest the hypothesis that SmTAL1 regulates at least one voltage-gated calcium channel and PZQ interferes with this process. This may be important in the molecular mechanism of this drug. It also suggests that compounds which bind SmTAL1, such as six from the Medicines for Malaria Box identified in this work, may represent possible leads for the discovery of novel antagonists.

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.

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.

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.

Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis. It is a potential drug target in the treatment of type II diabetes. The protein is also associated with a rare inherited metabolic disease and some cancer cells lack FBPase activity which promotes glycolysis facilitating the Warburg effect. Thus, there is interest in both inhibiting the enzyme (for diabetes treatment) and restoring its activity (in relevant cancers). The mammalian enzyme is tetrameric, competitively inhibited by Fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the active site of the enzyme. A recent paper by Topaz et al. (Bioscience Reports (2019) 39, pii:BSR20180960) has added additional detail to our understanding of this information transmission process. Two residues in the AMP binding site (Lys112 and Tyr113) were shown to be involved in initiating the message between the two sites. This tyrosine residue has recently be shown to be important with protein’s interaction with the antidiabetic drug metformin. A variant designed to increase metal ion affinity (M248D) resulted in a five-fold increase in enzymatic activity. Interestingly alterations of two residues at the subunit interfaces (Tyr164 and Met177) resulted in increased responsiveness to AMP. Overall, these findings may have implications in the design of novel FBPase inhibitors or activators.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease., This work was supported by the Junta de Andalucía [grant number P11-CTS-07187 (to A.L.P.)].

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.

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.

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.

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.

Schistosoma mansoni , like other trematodes, expresses a number of unusual calcium binding proteins which consist of an EF-hand domain joined to a dynein light chain-like (DLC-like) domain by a flexible linker. These proteins have been implicated in host immune responses and drug binding. Three members of this protein family from S. mansoni (SmTAL1, SmTAL2 and SmTAL3) have been well characterised biochemically. Here we characterise the remaining family members from this species (SmTAL4-13). All of these proteins form homodimers and all except SmTAL5 bind to calcium and manganese ions. SmTAL9, 10 and 11 also bind to magnesium ions. The antischistosomal drug, praziquantel interacts with SmTAL4, 5 and 8. Some family members also bind to calmodulin antagonists such as chlorpromazine and trifluoperazine. Molecular modelling suggests that all ten proteins adopt similar overall folds with the EF-hand and DLC-like domains folding discretely. Bioinformatics analyses suggest that the proteins may fall into two main categories: (i) those which bind calcium ions reversibly at the second EF-hand and may play a role in signalling (SmTAL1, 2, 8 and 12) and (ii) those which bind calcium ions at the first EF-hand and may play either signalling or structural roles (SmTAL7, 9, 10 and 13). The remaining proteins include those which do not bind calcium ions (SmTAL3 and 5) and three other proteins (SmTAL4, 6 and 11). The roles of these proteins are less clear, but they may also have structural roles.

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/β-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/β-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.

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.

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.

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)).

Citrate synthase catalyses the first step of the Krebs’ tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

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.

Galactokinase catalyses the site- and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C(1)-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the γ-phosphorus of ATP.

The 67LR (67 kDa laminin receptor) is a cell-surface receptor with high affinity for its primary ligand. Its role as a laminin receptor makes it an important molecule both in cell adhesion to the basement membrane and in signalling transduction following this binding event. The protein also plays critical roles in the metastasis of tumour cells. Isolation of the protein from either normal or cancerous cells results in a product with an approx. molecular mass of 67 kDa. This protein is believed to be derived from a smaller precursor, the 37LRP (37 kDa laminin receptor precursor). However, the precise mechanism by which cytoplasmic 37LRP becomes cell-membrane-embedded 67LR is unclear. The process may involve post-translational fatty acylation of the protein combined with either homo- or hetero-dimerization, possibly with a galectin-3-epitope-containing partner. Furthermore, it has become clear that acting as a receptor for laminin is not the only function of this protein. 67LR also acts as a receptor for viruses, such as Sindbis virus and dengue virus, and is involved with internalization of the prion protein. Interestingly, unmodified 37LRP is a ribosomal component and homologues of this protein are found in all five kingdoms. In addition, it appears to be strongly associated with histones in the eukaryotic cell nucleus, although the precise role of these interactions is not clear. Here we review the current understanding of the structure and function of this molecule, as well as highlighting areas requiring further research.

Metabolic pathways that extract energy from carbon compounds are essential for an organism's survival. Therefore, inhibition of enzymes in these pathways represents a potential therapeutic strategy to combat parasitic infections. However, the high degree of similarity between host and parasite enzymes makes this strategy potentially difficult. Nevertheless, several existing drugs to treat infections by parasitic helminths (worms) target metabolic enzymes. These include the trivalent antimonials that target phosphofructokinase and Clorsulon that targets phosphoglycerate mutase and phosphoglycerate kinase. Glycolytic enzymes from a variety of helminths have been characterised biochemically, and some inhibitors identified. To date none of these inhibitors have been developed into therapies. Many of these enzymes are externalised from the parasite and so are also of interest in the development of potential vaccines. Less work has been done on tricarboxylic acid cycle enzymes and oxidative phosphorylation complexes. Again, while some inhibitors have been identified none have been developed into drug-like molecules. Barriers to the development of novel drugs targeting metabolic enzymes include the lack of experimentally determined structures of helminth enzymes, lack of direct proof that the enzymes are vital in the parasites and lack of cell culture systems for many helminth species. Nevertheless, the success of Clorsulon (which discriminates between highly similar host and parasite enzymes) should inspire us to consider making serious efforts to discover novel anthelminthics, which target metabolic enzymes.

From in silico docking and COMPARE analysis, novel inhibitors of human NAD(P)H quinone oxidoreductase (NQO1) have been identified from the NCI compound database, the most potent of which has an observed IC(50) of 0.7muM. The inhibitors exhibit a diverse range of scaffolds. The ability of docking calculations to predict experimentally determined binding affinities for NQO1 is discussed, considering the influence of target flexibility and scoring function.

Type III galactosemia is a genetic disease caused by mutations in the gene encoding UDP-galactose 4-epimerase. A variety of different point mutations located throughout the gene can be responsible. The main, disease-causing effects of these mutations appear to be a reduction in the catalytic rate constant (kcat) and an increase in the proteolytic sensitivity of the protein. Many of the mutations are distant from the active site of the enzyme and therefore must be assumed to affect the overall fold of the protein. Although the disease was previously classified into a severe, or generalized, form and an essentially benign, or peripheral, form this distinction has been blurred by recent work. Instead of two separate conditions it now appears that type III galactosemia is a continuum and that the symptoms will vary depending on the mutation(s) carried by the individual sufferer. This new way of looking at the disease has implications for the treatment and long term monitoring of patients.

UDP-galactose 4-epimerase (GALE, EC 5.1.3.2) catalyses the interconversion of UDP-glucose and UDP-galactose. Point mutations in this enzyme are associated with the genetic disease, type III galactosemia, which exists in two forms – a milder, or peripheral, form and a more severe, or generalized, form. Recombinant wild-type GALE, and nine disease-causing mutations, have all been expressed in, and purified from, Escherichia coli in soluble, active forms. Two of the mutations (N34S and G319E) display essentially wild-type kinetics. The remainder (G90E, V94M, D103G, L183P, K257R, L313M and R335H) are all impaired in turnover number (kcat) and specificity constant (kcat/Km), with G90E and V94M (which is associated with the generalized form of galactosemia) being the most affected. None of the mutations results in a greater than threefold change in the Michaelis constant (Km). Protein–protein crosslinking suggests that none of the mutants are impaired in homodimer formation. The L183P mutation suffers from severe proteolytic degradation during expression and purification. N34S, G90E and D103G all show increased susceptibility to digestion in limited proteolysis experiments. Therefore, it is suggested that reduced catalytic efficiency and increased proteolytic susceptibility of GALE are causative factors in type III galactosemia. Furthermore, there is an approximate correlation between the severity of these defects in the protein structure and function, and the symptoms observed in patients.

Galactokinase functions in the Leloir pathway for galactose metabolism by catalyzing the MgATP-dependent phosphorylation of the C-1 hydroxyl group of alpha-D-galactose. The enzyme is known to belong to the GHMP superfamily of small molecule kinases and has attracted significant research attention for well over 40 years. Approximately 20 mutations have now been identified in human galactokinase, which result in the diseased state referred to as Type II galactosemia. Here we report the three-dimensional architecture of human galactokinase with bound alpha-D-galactose and Mg-AMPPNP. The overall fold of the molecule can be described in terms of two domains with the active site wedged between them. The N-terminal domain is dominated by a six-stranded mixed beta-sheet whereas the C-terminal motif contains six alpha-helices and two layers of anti-parallel beta-sheet. Those residues specifically involved in sugar binding include Arg37, Glu43, His44, Asp46, Gly183, Asp186, and Tyr236. The C-1 hydroxyl group of alpha-D-galactose sits within 3.3 A of the gamma-phosphorus of the nucleotide and 3.4 A of the guanidinium group of Arg37. The carboxylate side chain of Asp186 lies within approximately 3.2 A of the C-2 hydroxyl group of alpha-D-galactose and the guanidinium group of Arg37. Both Arg37 and Asp186 are strictly conserved among both prokaryotic and eukaryotic galactokinases. In addition to providing molecular insight into the active site geometry of the enzyme, the model also provides a structural framework upon which to more fully understand the consequences of the those mutations known to give rise to Type II galactosemia.

Galactose mutarotase catalyzes the conversion of beta-d-galactose to alpha-d-galactose during normal galactose metabolism. The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells. Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, beta-d-galactose. The polypeptide chain folds into an intricate array of 29 beta-strands, 25 classical reverse turns, and 2 small alpha-helices. There are two cis-peptide bonds at Arg-78 and Pro-103. The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307. Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively. These residues are absolutely conserved among galactose mutarotases. To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans. The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two beta-strands. In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 A. In the C. elegans protein, the first 17 residues are missing, thereby reducing the total number of beta-strands by one.

Galactokinase (GaIK) catalyses the first step of the Leloir pathway of galactose metabolism, the ATP-dependent phosphorylation of galactose to galactose-l-phosphate. In man, defects in galactose metabolism can result in disorders with severe clinical consequences, and deficiencies in galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus furiosus in complex with MgADP and galactose has been determined to 2.9 Angstrom resolution to provide insights into the substrate specificity and catalytic mechanism of the enzyme. The structure consists of two domains with the active site in a cleft at the domain interface. Inspection of the substrate binding pocket identifies the amino acid residues involved in galactose and nucleotide binding and points to both structural and mechanistic similarities with other enzymes of the GHMP kinase superfamily to which GalK belongs. Comparison of the sequence of the Gal3p inducer protein, which is related to GalK and which forms part of the transcriptional activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, has led to an understanding of the molecular basis of galactose and nucleotide recognition. Finally, the structure has enabled us to further our understanding on the functional consequences of mutations in human GalK which cause galactosemia. (C) 2004 Elsevier Ltd. All rights reserved.

Aldose 1-epimerase or mutarotase (EC 5.1.3.3) is a key enzyme of carbohydrate metabolism catalysing the interconversion of the α- and β-anomers of hexose sugars such as glucose and galactose. We identified an open reading frame in the human genome (BC014916) which has high sequence similarity to previously identified bacterial aldose 1-epimerases. This sequence was cloned into a bacterial expression vector, and expressed and purified from this source. Enzyme assays show that the protein has aldose 1-epimerase activity and exhibits a preference for galactose over glucose. Site-directed mutagenesis confirmed the involvement of three residues involved in catalysis and substrate binding.

The genes encoding the enzymes required for galactose metabolism in Saccharomyces cerevisiae are controlled at the level of transcription by a genetic switch consisting of three proteins: a transcriptional activator, Gal4p; a transcriptional repressor, Gal80p; and a ligand sensor, Gal3p. The switch is turned on in the presence of two small molecule ligands, galactose and ATP. Gal3p shows a high degree of sequence identity with Gal1p, the yeast galactokinase. We have mapped the interaction between Gal80p and Gal3p, which only occurs in the presence of both ligands, using protease protection experiments and have shown that this involves amino acid residue 331 of Gal80p. Gel-filtration experiments indicate that Gal3p, or the galactokinase Gal1p, interact directly with Gal80p to form a complex with 1:1 stoichiometry.

Highlights • SNP rs1143684 results in either a Phe or Leu at position 47 in human NQO2. • NQO2-L47 has a slightly lower kcat/Km; it also has a lower Ki,app with resveratrol. • NQO2-L47 is more unstable to proteolysis and thermal denaturation. • NQO2-F47 (but not NQO2-L47) shows negative cooperativity towards resveratrol. • NQO2-L47 exists as multiple conformers in solution., There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings.

Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization.

DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA. Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD+. Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.

The gene for DNA ligase (EC 6.5.1.2) from thermophilic bacterium Bacillus stearothermophilus NCA1503 has been cloned and the complete nucleotide sequence determined. The ligase gene encodes a protein 670 amino acids in length. The gene was overexpressed in Escherichia coli and the enzyme has been purified to homogeneity. Preliminary characterisation confirms that it is a thermostable, NAD + -dependent DNA ligase.

The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by 1 H NMR that the major actin binding region occurred in the N-terminal four residues, APKK .... These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys 3 or Lys 4 with Ala resulted in increased Km and kcat and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys 4 with Asp reduced actin binding and increased Km and kcat still further. Alteration of Ala 1 to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain’s ability to bind actin. Furthermore, we found a significant correlation between the apparent Km for actin and the kcat for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.

The crystal structure of an ATP-dependent DNA ligase from bacteriophage T7 revealed that the protein comprised two structural domains. In order to investigate the biochemical activities of these domains, we have overexpressed them separately and purified them to homogeneity. The larger N-terminal domain retains adenylation and ligase activities, though both at a reduced level. The adenylation activity of the large domain is stimulated by the presence of the smaller domain, suggesting that a conformational change is required for adenylation in the full length protein. The DNA binding properties of the two fragments have also been studied. The larger domain is able to band shift both single and double-stranded DNA, while the smaller fragment is only able to bind to double-stranded DNA. These data suggest that the specificity of DNA ligases for nick sites in DNA is produced by a combination of these different DNA binding activities in the intact enzyme.

Background: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD + 4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target. Results: The crystal structure of the adenylation domain of the NAD + -dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 A resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD + -binding site. Conclusions: Comparison of the structure of the NAD + 4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.

We have shown that Fasciola hepatica expresses at least six β-tubulins in the adult stage of its life cycle, designated F.hep-β-tub1–6 (Ryan et al., 2008). Here we show that different complements of tubulin isotypes are expressed in different tissues and at different life cycle stages; this information may inform the search for novel anthelmintics. The predominant (as judged by quantitative PCR) isotype transcribed at the adult stage was F.hep-β-tub1 and immunolocalisation studies revealed that this isotype occurred mainly in mature spermatozoa and vitelline follicles. Quantitative PCR indicated that changes occurred in the transcription levels of β-tubulin isotypes at certain life cycle stages and may be of importance in the efficacy of benzimidazole-based anthelmintic drugs, but there were no significant differences between the triclabendazole-susceptible Leon isolate and the triclabendazole-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to triclabendazole were compared, only one amino acid change resulting from a homozygous coding sequence difference (Gly269Ser) in isotype 4 was observed. However, this change was not predicted to alter the overall structure of the protein. In conclusion, these findings indicate that there is tissue-specific expression of tubulin isotypes in the liver fluke but the development of resistance to triclabendazole is not associated with changes in its presumed target molecule.

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a β-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis-Menten kinetics in both directions, with Km values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s(-1) for the conversion of dihydroxyacetone phosphate and 1900 s(-1) for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. β-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell’s macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme β-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V max, to fall and the Michaelis constant, K m, to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited β-galactosidase, the addition of compatible solutes was unable to restore the enzyme’s kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme’s structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.

IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and micro- tubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recom- binant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium-calmod- ulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interac- tion remains to be discovered.

Laser desorption of dye-tagged oligonucleotides was studied using laser-induced fluorescence imaging. Desorption with ultra violet (UV) and infra-red (IR) lasers resulted in forward directed plumes of molecules. In the case of UV desorption, the initial shot desorbed approximately seven-fold more material than subsequent shots. In contrast, the initial shot in IR desorption resulted in the ejection of less material compared to subsequent shots and these plumes had a component directed along the path of the laser. Thermal equilibrium of the molecules in the plume was achieved after approximately 25 μs with a spread in molecular temperature which was described by a modified Maxwell–Boltzmann equation.

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins.

N-acetylgalactosamine kinase is a member of the GHMP family of small molecule kinases which catalyses the ATP-dependent phosphorylation of N-acetylgalactosamine. It is highly similar in structure and sequence to galactokinase. Alteration of galactokinase at a key tyrosine residue (Tyr-379 in the human enzyme) has been shown to dramatically enhance the substrate range of this enzyme. Here, we investigated the substrate specificity of the wild type N-acetylgalactosamine kinase and demonstrated that it can also catalyse the phosphorylation of N-acetylglucosamine and N-acetylmannosamine. In human N-acetylgalactosamine kinase, the equivalent residue to Tyr-379 in galactokinase is Phe-444. Alteration of this residue did not result in dramatic changes to the specificity of the enzyme. The more relaxed substrate specificity of N-acetylgalactosamine kinase, compared to galactokinase, can be explained by the greater flexibility of a glycine rich loop in the active site of the enzyme. These results suggest that N-acetylgalactosamine kinase is a potential biocatalyst for the phosphorylation of N-acetyl sugars. However, it is unlikely that it will be possible to further broaden the substrate range by alteration of Phe-444.

Type I galactosemia results from reduced galactose 1-phosphate uridylyltransferase (GALT) activity. Signs of disease include damage to the eyes, brain, liver, and ovaries. However, the exact nature and severity of the pathology depends on the mutation(s) in the patient's genes and his/her environment. Considerable enzymological and structural knowledge has been accumulated and this provides a basis to explain, at a biochemical level, impairment in the enzyme in the more than 230 disease-associated variants, which have been described. The most common variant, Q188R, occurs close to the active site and the dimer interface. The substitution probably disrupts both UDP-sugar binding and homodimer stability. Other alterations, for example K285N, occur close to the surface of the enzyme and most likely affect the folding and stability of the enzyme. There are a number of unanswered questions in the field, which require resolution. These include the possibility that the main enzymes of galactose metabolism form a supramolecular complex and the need for a high resolution crystal structure of human GALT.