New Insights to Regulation of Fructose-1,6-bisphosphatase during Anoxia in Red-Eared Slider, Trachemys scripta elegans.

The red-eared slider (Trachemys scripta elegans) undergoes numerous changes to its physiological and metabolic processes to survive without oxygen. During anoxic conditions, its metabolic rate drops drastically to minimize energy requirements. The alterations in the central metabolic pathways are often accomplished by the regulation of key enzymes. The regulation of one such enzyme, fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), was characterized in the present study during anoxia in liver. FBPase is a crucial enzyme of gluconeogenesis. The FBPase was purified from liver tissue in both control and anoxic conditions and subsequently assayed to determine the kinetic parameters of the enzyme. The study revealed the relative degree of post-translational modifications in the FBPase from control and anoxic turtles. Further, this study demonstrated a significant decrease in the maximal activity in anoxic FBPase and decreased sensitivity to its substrate Fructose-1,6-bisphosphate (FBP) when compared to the control. Immunoblotting demonstrated increased threonine phosphorylation (~1.4-fold) in the anoxic FBPase. Taken together, these results suggest that the phosphorylation of liver FBPase is an important step in suppressing FBPase activity, ultimately leading to the inhibition of gluconeogenesis in the liver of the red-eared slider during anaerobic conditions.

Efficient biosynthesis of D-allulose in Bacillus subtilis through D-psicose 3-epimerase translation modification.

The combined catalysis of glucose isomerase (GI) and D-psicose 3-epimerase (DPEase) provided a convenient route for the direct synthesis of D-allulose from d-glucose, whose cost is lower than d-fructose. In the present research, the weak activity of DPEase was the key rate-limiting step and resulted in the accumulation of d-fructose in engineered Bacillus subtilis. Then, the 5'-untranslated region (5'-UTR) structure of the mRNA translational initiation region was optimized for the precise control of DPEase expression. The manipulation of the 5'-UTR region promoted the accessibility to ribosome binding and the stability of mRNA, resulting in a maximum of 1.73- and 1.98-fold increase in DPEase activity and intracellular mRNA amount, respectively. Under the optimal catalytic conditions of 75 °C, pH 6.5, 110 g/L d-glucose, and 1 mmol/L Co2+, the reaction equilibrium time was reduced from 7.6 h to 6.1 h. We hope that our results could provide a facilitated strategy for large-scale production of D-allulose at low-cost.

Characterization of a Recombinant D-Allulose 3-epimerase from Thermoclostridium caenicola with Potential Application in D-Allulose Production.

In recent years, with the increasing public health awareness, low-calorie rare sugars have received more attention on a global scale. D-Allulose, the C-3 epimer of D-fructose, is a representative rare sugar. It displays high sweetness and excellent physiological functions, but only provides a caloric value of 0.4 kcal/g. D-Allulose 3-epimerase (DAEase) is indispensable in D-allulose production. In this study, a putative DAEase from Thermoclostridium caenicola was identified and characterized. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 °C in the presence of 1 mM Co2+. The half-life (t1/2) at 50 °C was 13.6 h, and the melting temperature (Tm) was 62.4 °C. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t1/2 value at 55 °C and 4.8 °C increase in Tm. Furthermore, DAEase displayed high relative activity (89.0%) at a weakly acidic pH 6.5 and produced 139.8 g/L D-allulose from 500 g/L D-fructose, achieving a conversion ratio of 28.0%. These findings suggest that T. caenicola DAEase is a promising biocatalyst for the production of D-allulose.

A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1,6-Bisphosphate Aldolase A.

Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z' (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.

Genome-wide characterization of the NUCLEAR FACTOR-Y (NF-Y) family in Citrus grandis identified CgNF-YB9 involved in the fructose and glucose accumulation.

BACKGROUND: Nuclear factor Y (NF-Y) is increasingly known to be involved in many aspects of plant growth and development. To date, the systematic characterization of NF-Y family has never been reported in Citrus grandis. OBJECTIVE: Genome-wide characterization of C. grandis NF-Y (CgNF-Y) family and analysis of their role in sucrose metabolism. METHODS: NF-Y conserved models were employed to identify CgNF-Y genes from genomic data. Phylogenetic tree was generated by the neighbor-joining method using program MEGA 7.0. Based on our previous transcriptomic data, the transcription levels were calculated by RSEM software and were clustered by ShortTime-series Expression Miner. The plant expression vector of CgNF-YB9 was constructed using In-Fusion Cloning and transferred into tobacco by leaf disc transformation method. Soluble sugars and gene expressions were analysis by HPLC and qRT-PCR, respectively. RESULTS: A total of 24 CgNF-Y genes (6 CgNF-YAs, 13 CgNF-YBs and 5 CgNF-YCs) were identified with conserved domains. Phylogenetic analysis of the NF-Y proteins indicated that NF-YA, NF-YB and NF-YC could be categorized into four, five and three clades, respectively. Expression profiling analysis reflected spatio-temporally distinct expression patterns for CgNF-Y genes. Importantly, we observed a positive correlation between the expression level of CgNF-YB9 and the content of soluble sugar. Moreover, CgNF-YB9-corelated genes were enriched in carbohydrate metabolism. In CgNF-YB9 overexpression lines, sucrose content showed a decrease, whereas glucose and fructose contents displayed an increase. As expected, the transcription levels of sucrose-phosphate synthase and vacuolar invertase in transgenic Line 3 were observed with significantly down- and up-regulated, respectively. CONCLUSIONS: The structure, phylogenetic relationship and expression pattern of 24 CgNF-Y genes were identified, and CgNF-YB9 was involved in sucrose metabolism.

Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampal function in offspring, indicating that the hippocampi of offspring are highly sensitive to maternal fructose. Here, we examined the effect of maternal high fructose on mitochondrial physiology and uncoupling protein (UCP) expression. Rat dams received a 20% fructose solution during gestation and lactation. Immediately after weaning, offspring hippocampi were isolated. Maternal high fructose consumption attenuated the mitochondrial O2 consumption rate and stimulated lipid hydroperoxide production in the hippocampi of offspring. Reduced Ucp5 and mitochondrial transcription factor A (Tfam) mRNA levels were also observed after maternal exposure to fructose. We assessed the promoter regions of both genes and found that this treatment enhanced DNA methylation levels. In addition, luciferase assays showed that this DNA methylation could reduce the transcription of both genes. Chromatin immunoprecipitation analysis demonstrated that specificity protein 1 binding to the Ucp5 promoter regions was reduced by DNA methylation. In addition, Ucp5 knockdown induced the up-regulation of reactive oxygen species levels in a rat brain glioma cell line, whereas reduced O2 consumption was observed with Tfam knockdown. Maternal high fructose intake thus induces reduced O2 oxygen consumption and increases oxidative stress in offspring, at least partly through epigenetic mechanisms involving Ucp5 and Tfam.-Yamada, H., Munetsuna, E., Yamazaki, M., Mizuno, G., Sadamoto, N., Ando, Y., Fujii, R., Shiogama, K., Ishikawa, H., Suzuki, K., Shimono, Y., Ohashi, K., Hashimoto, S. Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

The relevance of ANXA5 genetic variants on male fertility.

PURPOSE: To investigate the effect of the anticoagulation factor annexin A5 on male fertility and to provide perspective on the influence of members of the coagulation cascade on fertility. METHODS: Patients with normozoospermia and with unexplained severe oligozoospermia were retrospectively selected and their genomic DNA sequenced for the promoter region of ANXA5. The genotypes proportions and the odds ratio for carriership of the haplotype M2 were compared between the groups and population control. The clinical data used were gathered from parameters determined during routine clinical assessment and were compared between carriers and non-carriers within the patient groups. RESULTS: The carrier rates for the haplotype M2/ANXA5 were of 25.73%, 20.81%, and 15.3% in the severe oligozoospermic, the normozoospermic, and the general population control groups, respectively. The OR between patients groups was of 1.31 (95% CI 0.88 to 1.96 p = 0.176). Oligozoospermic and normozoospermic patients compared with the control group had an OR of 1.9 (95% CI 1.33 to 2.73 p < 0.001) and 1.45 (95% CI 0.99 to 2.10 p = 0.054) respectively. The clinical parameters that differed between the carriers and non-carriers of the haplotype M2/ANXA5 were prolactin, α-glucosidase, and fructose. The differences were only statistically significant in the normozoospermic group. CONCLUSIONS: Athough the infertile patient groups had a higher prevalence of promoter variants, we could not demonstrate any biologically relevant effect of lower levels of annexin A5 on most male fertility parameters. A deficiency in an anticoagulation factor does not seem to impact male fertility.

Meta-analysis of genome-wide association studies provides insights into genetic control of tomato flavor.

Tomato flavor has changed over the course of long-term domestication and intensive breeding. To understand the genetic control of flavor, we report the meta-analysis of genome-wide association studies (GWAS) using 775 tomato accessions and 2,316,117 SNPs from three GWAS panels. We discover 305 significant associations for the contents of sugars, acids, amino acids, and flavor-related volatiles. We demonstrate that fruit citrate and malate contents have been impacted by selection during domestication and improvement, while sugar content has undergone less stringent selection. We suggest that it may be possible to significantly increase volatiles that positively contribute to consumer preferences while reducing unpleasant volatiles, by selection of the relevant allele combinations. Our results provide genetic insights into the influence of human selection on tomato flavor and demonstrate the benefits obtained from meta-analysis.

Transcriptome signature for dietary fructose-specific changes in rat renal cortex: A quantitative approach to physiological relevance.

Fructose consumption causes metabolic diseases and renal injury primarily in the renal cortex where fructose is metabolized. Analyzing gene differential expression induced by dietary manipulation is challenging. The effects may depend on the base diet and primary changes likely induce secondary or higher order changes that are difficult to capture by conventional univariate transcriptome analyses. We hypothesized that dietary fructose induces a genetic program in the kidney cortex that favors lipogenesis and gluconeogenesis. To test this, we analyzed renal cortical transcriptomes of rats on normal- and high-salt base diets supplemented with fructose. Both sets of data were analyzed using the Characteristic Direction method to yield fructose-induced gene vectors of associated differential expression values. A fructose-specific "signature" of 139 genes differentially expressed was extracted from the 2 diet vectors by a new algorithm that takes into account a gene's rank and standard deviation of its differential expression value. Of these genes, 97 were annotated and the top 34 accounted for 80% of the signal in the annotated signature. The genes were predominantly proximal tubule-specific, coding for metabolic enzymes or transporters. Cosine similarity of signature genes in the two fructose-induced vectors was >0.78. These 139 genes of the fructose signature contributed 27% and 38% of total differential expression on normal- and high- salt diet, respectively. Principal Component Analysis showed that the individual animals could be grouped according to diet. The fructose signature contained a greater enrichment of Gene Ontology processes related to nutrition and metabolism of fructose than two univariate analysis methods. The major feature of the fructose signature is a change in metabolic programs of the renal proximal tubule consistent with gluconeogenesis and de-novo lipogenesis. This new "signature" constitutes a new metric to bridge the gap between physiological phenomena and differential expression profile.

Insights into hydrolysis versus transfructosylation: Mutagenesis studies of a novel levansucrase from Brenneria sp. EniD312.

Levan is a kind of fructan that composing of fructose by ß-(2, 6) linkage and has been already applied as thickening agent and colloidal stabilizer in the cosmetic, medicinal and food industries. Microbial levansucrase is a key enzyme catalyzing the formation of levan from sucrose by transfructosylation. Here, a gene encoding levansucrase from Brenneria sp. EniD312 was cloned and expressed in Escherichia coli. The recombinant levansucrase showed the optimal pH and temperature at pH 6.5 and 45 °C. The enzyme produced 85 g/L levan from 250 g/L sucrose at pH 6.5 and 45 °C for 6 h. The residues D68, D225 and E309 of this levansucrase were speculated to be the nucleophile, the transition stabilizer and the general acid respectively by homology modelling, site-directed mutagenesis and molecular docking. Particularly, the residues in position 154 and 327 were found to play a significant role in determining the ratio of hydrolysis activity to transfructosylation activity.

Ketohexokinase C blockade ameliorates fructose-induced metabolic dysfunction in fructose-sensitive mice.

Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of aldolase B results in the accumulation of intracellular phosphorylated fructose, leading to phosphate sequestration and depletion, increased adenosine triphosphate (ATP) turnover, and a plethora of conditions that lead to clinical manifestations such as fatty liver, hyperuricemia, Fanconi syndrome, and severe hypoglycemia. Unfortunately, there is currently no treatment for HFI, and avoiding sugar and fructose has become challenging in our society. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI. Herein we provide evidence for the first time to our knowledge of a potential therapeutic approach for HFI. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction.

Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd.

Glucose transporter 8 (GLUT8) mediates fructose-induced de novo lipogenesis and macrosteatosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αßα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

The structure of substrate-free 1,5-anhydro-D-fructose reductase from Sinorhizobium meliloti 1021 reveals an open enzyme conformation.

1,5-Anhydro-D-fructose (1,5-AF) is an interesting building block for enantioselective and stereoselective organic synthesis. Enzymes acting on this compound are potential targets for structure-based protein/enzyme design to extend the repertoire of catalytic modifications of this and related building blocks. Recombinant 1,5-anhydro-D-fructose reductase (AFR) from Sinorhizobium meliloti 1021 was produced in Escherichia coli, purified using a fused 6×His affinity tag and crystallized in complex with the cofactor NADP(H) using the hanging-drop technique. Its structure was determined to 1.93 Å resolution using molecular replacement. The structure displays an empty substrate-binding site and can be interpreted as an open conformation reflecting the enzyme state shortly after the release of product, presumably with bound oxidized cofactor NADP⁺. Docking simulations indicated that amino-acid residues Lys94, His151, Trp162, Arg163, Asp176 and His180 are involved in substrate binding, catalysis or product release. The side chain of Lys94 seems to have the ability to function as a molecular switch. The crystal structure helps to characterize the interface relevant for dimer formation as observed in solution. The crystal structure is compared with the structure of the homologue from S. morelense, which was solved in a closed conformation and for which dimer formation in solution could not be verified but seems to be likely based on the presented studies of S. meliloti AFR.

Structural mechanism of ring-opening reaction of glucose by human serum albumin.

Glucose reacts with proteins nonenzymatically under physiological conditions. Such glycation is exacerbated in diabetic patients with high levels of blood sugar and induces various complications. Human albumin serum (HSA) is the most abundant protein in plasma and is glycated by glucose. The glycation sites on HSA remain controversial among different studies. Here, we report two protein crystal structures of HSA in complex with either glucose or fructose. These crystal structures reveal the presence of linear forms of sugar for both monosaccharides. The linear form of glucose forms a covalent bond to Lys-195 of HSA, but this is not the case for fructose. Based on these structures, we propose a mechanism for glucose ring opening involving both residues Lys-195 and Lys-199. These results provide mechanistic insights to understand the glucose ring-opening reaction and the glycation of proteins by monosaccharides.

The post-illumination chlorophyll fluorescence transient indicates the RuBP regeneration limitation of photosynthesis in low light in Arabidopsis.

The mechanism of post-illumination chlorophyll fluorescence transient (PIFT) was investigated in Arabidopsis. PIFT was detected in the wild type after illumination with low light. In the fba3-2 (fructose-1,6-bisphosphate aldolase) mutant, in which PIFT is enhanced, strong light also induced PIFT. PIFT was suppressed not only in the triose phosphate/phosphate translocator (tpt-2) mutant, but also in tpt-2 fba3-2, suggesting that triose phosphates, such as dihydroxyacetone phosphate (DHAP), are involved in the PIFT mechanism. We concluded that PIFT is associated with ribulose-1,5-bisphosphate (RuBP)-regeneration limitation of photosynthesis in low light.

Structural and kinetic analysis of Schwanniomyces occidentalis invertase reveals a new oligomerization pattern and the role of its supplementary domain in substrate binding.

Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolyzes sucrose and releases beta-fructose from various oligosaccharides and essential storage fructan polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 A resolution and its complex with fructose at 1.9 A resolution. The monomer presents a bimodular arrangement common to other GH32 enzymes, with an N-terminal 5-fold beta-propeller catalytic domain and a C-terminal beta-sandwich domain for which the function has been unknown until now. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin was explored by docking analysis, which suggested that medium size and long substrates are recognized by residues from both subunits. The identified residues were mutated, and the enzymatic activity of the mutants against sucrose, nystose, and inulin were investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency were Q228V, a residue putatively involved in nystose and inulin binding, and S281I, involved in a polar link at the dimer interface. Moreover, a significant decrease in catalytic efficiency against inulin was observed in the mutants Q435A and Y462A, both located in the beta-sandwich domain of the second monomer. This highlights the essential function that oligomerization plays in substrate specificity and assigns, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme.

Does an apple a day put hypertension in play?

Singh et al. offer exciting data in their new report about fructose-induced hypertension. Fructose induced an increase in serum uric acid after 8-10 weeks of increased fructose exposure, but no correlation was found between hyperuricemia and hypertension.