Hexose Kinases Research Articles

Sugar composition and transcriptome analysis in developing ‘Fengtang’ plum (Prunus salicina Lindl.) reveal candidate genes regulating sugar accumulation

Sweetness is an important attribute of fruit quality, which directly affects consumers' preference for fresh fruit and is mostly determined by carbohydrate composition. ‘Fengtang’ plum (Prunus salicina Lindl.) is recognized for its high soluble sugar content, but the sugar composition and the molecular mechanisms underlying sugar overproduction are not fully understood. In this work, the sugar components were analyzed using gas chromatography-mass spectrometry combined with transcription profiles from RNA-sequencing and Quantitative Real-time PCR during fruit development. The target metabolic group showed that sucrose was the dominant sugar component in mature fruit, followed by glucose, fructose, and sorbitol. Based on the transcriptome data and qRT-PCR validation, we identified 12 key structural genes that significantly responded to corresponding component accumulation: sucrose synthase (PsSUS4), sucrose phosphate synthase (PsSPS2), neutral invertase (PsNINV1/3/4), phosphoglucomutase (PsPGM1), UTP-glucose-1-phosphate uridylyl transferase (PsUGP1/2), hexose kinase (PsHXK1/3), sugar transport protein (PsSTP1), and Sugars Will Eventually be Exported Transporter (PsSWEET4). In which PsSUS4 and PsSPS2, whose encoding proteins immediately catalyze sucrose synthesis, were selected to be silenced using the virus-induced gene silencing technology. Silencing of PsSUS4 or PsSPS2 resulted in decreased sucrose content by 27.6% and 8%, respectively, compared with the control, verifying their important roles in sucrose accumulation. Subsequently, sugar metabolism networks in this high-sugar plum were constructed with 12 key structural genes, 72 putative transcription factors, and 4 major sugar components. These results might facilitate a better understanding of the molecular mechanisms of sugar accumulation in ‘Fengtang’ plum and provide a framework for future fruit quality improvement.

VDAC upregulation and αTAT1‑mediated α‑tubulin acetylation contribute to tanespimycin‑induced apoptosis in Calu‑1 cells.

Voltage‑dependent anion channel 1 (VDAC1) functions as a porin in the mitochondrial outer membrane (MOM) and plays important roles in mitochondria‑mediated cell apoptosis. VDAC1 interacts with a variety of proteins, such as Bcl‑2 family proteins, hexose kinase (HK), adenine nucleotide translocase (ANT) and α‑tubulin. However, the association between VDAC1 and α‑tubulin, particularly between VDAC1 and acetylated α‑tubulin (Ac‑α‑tubulin), in apoptosis remains unclear. The present study revealed that the heat shock protein 90 inhibitor, tanespimycin, induced VDAC1 upregulation and α‑tubulin acetylation during Calu‑1 cell apoptosis in human lung cancer. Hsp90 mediated the expression level of VDAC1, and the acetylation of α‑tubulin was enhanced in an α‑tubulin acetyltransferase 1 (αTAT1)‑dependent manner following an increase in VDAC1 expression. Docetaxel, as an inhibitor of microtubules, augmented the expression of Ac‑α‑tubulin, VDAC1 and Bax induced by tanespimycin and increased the degree of caspase activation. Immunoprecipitation (IP) experiments revealed that Ac‑α‑tubulin, α‑tubulin and VDAC1 were co‑precipitated in the IP complex, in which α‑tubulin expression was decreased and VDAC1 proteins were oligomerized, and that the p‑AKT/glycogen synthase kinase 3β (GSK3β) signalling pathway mediated the opening of VDAC1. Therefore, it can be asserted that the acetylation of α‑tubulin and VDAC1 upregulation or oligomerization induced by tanespimycin may lead to mitochondrial permeability and consequently induce the apoptosis of lung cancer cells. These findings provide evidence for the use of a combination of drugs that target VDAC1 and tubulin to induce tumour cell apoptosis.

Mig1 localization exhibits biphasic behavior which is controlled by both metabolic and regulatory roles of the sugar kinases

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.

Regulation of glucose and lipid metabolism by insulin and glucagon in vivo and in vitro in common carp Cyprinus carpio L.

Our objective is to study the low metabolic utilization of dietary carbohydrates in fish. The regulation of key enzymes involved in glucose and lipid metabolism in the common carp (Cyprinus carpio L.), known to be relatively tolerant to dietary carbohydrates, were assessed. We performed intraperitoneal (i.p.) administration of insulin and glucagon to common carp and also subjected common carp primary hepatocytes to insulin or glucagon. The results showed that insulin administration induced hypoglycaemia through up-regulation of the expression of the glycolysis genes glucokinase (GK) and hexose kinase (HK), coupled with increased gene expression of fatty acid synthase (FAS), acetyl-CoA carboxylase-1 (ACC-1), sterol-regulator element-binding protein-1 (SREBP-1). Insulin also limited the expression of the gluconeogenesis genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), coupled with decreased gene expression of lipoprotein lipase (LPL) and carnitine palmitoyl transferase I (CPT-1). However, glucagon had the opposite effect. The results showed that glucagon induced hyperglycemia with increased expression of PEPCK and G6Pase and increased lipolytic capacity as indicated by lower mRNA levels of SREBP-1. Glucagon (i.p.) significantly increased the activity of PEPCK and LPL in hepatopancreas. In addition, the mRNA expression of HK showed an up-regulated trend and the mRNA expression of PEPCK and G6Pase showed a down-regulated trend in the group of glucose (i.p.). This study demonstrates that insulin and glucagon possess the intrinsic ability to regulate hepatic energy metabolism in common carp, the regulation of which seems to be similar to that of the mammals at least at the molecular level.

Hexokinase and Glucokinases Are Essential for Fitness and Virulence in the Pathogenic Yeast Candida albicans.

The pathogenic yeast Candida albicans is both a powerful commensal and a pathogen of humans that can infect wide range of organs and body sites. Metabolic flexibility promotes infection and commensal colonization by this opportunistic pathogen. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low levels in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient detection and metabolism are fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase (CaHxk2) and two glucokinases (CaGlk1 and CaGlk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly performed by CaHxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, CaHxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, CaHxk2 participates in oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans. By contributing to metabolic flexibility, stress response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans.

Blockade of glycolysis-dependent contraction by oroxylin a via inhibition of lactate dehydrogenase-a in hepatic stellate cells

BackgroundContraction of hepatic stellate cells (HSCs) plays an important role in the pathogenesis of liver fibrosis by regulating sinusoidal blood flow and extracellular matrix remodeling. Here, we investigated how HSC contraction was affected by the natural compound oroxylin A, and elucidated the underlying mechanism.MethodsCell contraction and glycolysis were examined in cultured human HSCs and mouse liver fibrosis model upon oroxylin A intervention using diversified cellular and molecular assays, as well as genetic approaches.ResultsOroxylin A limited HSC contraction associated with inhibiting myosin light chain 2 phosphorylation. Oroxylin A blocked aerobic glycolysis in HSCs evidenced by reduction in glucose uptake and consumption and lactate production. Oroxylin A also decreased extracellular acidification rate and inhibited the expression and activity of glycolysis rate-limiting enzymes (hexose kinase 2, phosphofructokinase 1 and pyruvate kinas type M2) in HSCs. Then, we identified that oroxylin A blockade of aerobic glycolysis contributed to inhibition of HSC contraction. Furthermore, oroxylin A inhibited the expression and activity of lactate dehydrogenase-A (LDH-A) in HSCs, which was required for oroxylin A blockade of glycolysis and suppression of contraction. Oral administration of oroxylin A at 40 mg/kg reduced liver injury and fibrosis, and inhibited HSC glycolysis and contraction in mice with carbon tetrachloride-induced hepatic fibrosis. However, adenovirus-mediated overexpression of LDH-A significantly counteracted the oroxylin A’s effects in fibrotic mice.ConclusionsBlockade of aerobic glycolysis by oroxylin A via inhibition of LDH-A reduced HSC contraction and attenuated liver fibrosis, suggesting LDH-A as a promising target for intervention of hepatic fibrosis.

Isolation and expression features of hexose kinase genes under various abiotic stresses in the tea plant (Camellia sinensis)

Hexokinases (HXKs, EC 2.7.1.1) and fructokinases (FRKs, EC 2.7.1.4) play important roles in carbohydrate metabolism and sugar signaling during the growth and development of plants. However, the HXKs and FRKs in the tea plant (Camellia sinensis) remain largely unknown. In this manuscript, we present the molecular characterization, phylogenetic relationships, conserved domains and expression profiles of four HXK and seven FRK genes of the tea plant. The 11 deduced CsHXK and CsFRK proteins were grouped into six main classes. All of the deduced proteins, except for CsFKR7, possessed putative ATP-binding motifs and a sugar recognition region. These genes exhibited tissue-specific expression patterns, which suggests that they play different roles in the metabolism and development of source and sink tissues in the tea plant. There were variations in CsHXKs and CsFRKs transcript abundance in response to four abiotic stresses: cold, salt, drought and exogenous abscisic acid (ABA). Remarkably, CsHXK3 and CsHXK4 were significantly induced in the leaves and roots under cold conditions, CsHXK1 was apparently up-regulated in the leaves and roots under salt and drought stresses, and CsHXK3 was obviously stimulated in the leaves and roots under short-term treatment with exogenous ABA. These findings demonstrate that CsHXKs play critical roles in response to abiotic stresses in the tea plant. Our research provides a fundamental understanding of the CsHXK and CsFRK genes of the tea plant and important information for the breeding of stress-tolerant tea cultivars.

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids.

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast.

Reliability of bedside blood glucose estimating methods in detecting hypoglycaemia in the children’s emergency room

Background: Hypoglycaemia occurs in many disease states common in the tropics. Facilities and skilled manpower required for laboratory blood glucose measurement are not always available in health facilities in developing countries. Objective: The study was carried out to determine the validity of bedside methods of blood glucose measurement in detecting hypoglycaemia.Methods: Blood glucose was determined by two bedside methods (Accuchek Active® and Betachek Visual®) in 430 patients aged between one month and 10years and simultaneously sent for laboratory spectrophotometric analysis at a wavelength of 500nm using the hexose kinase method. Hypoglycaemia was defined as plasma glucose < 2.5mmol/L.Results: The prevalence of hypoglycaemia was 5.6%.There was a higher correlation (r =0.84, p< 0.05) between Accuchek Active® results and laboratory values than was obtained with Betachek Visual ® (r = 0.48, p=0.000).In detection of hypoglycaemia, both bedside glucose monitors were found to have a high specificity and high predictive values of a negative test (99.8% and 98.5% for Accuchek Active® and 89.4% and 97.8% for Betachek Visual® respectively) with moderate sensitivity (75.0% and 66.7% respectively). However, the Accuchek Active® monitor has a much higher predictive value of a positive test (94.1%) compared to the Betachek Visual® (27.1%).Conclusion: The bedside glucose monitors are valid bedside tools for detecting or ruling out hypoglycemia.Keywords: hypoglycaemia, children, bedside investigation, glucose estimation, blood glucose.

Evolution of moonlighting proteins: insight from yeasts.

The present article addresses the possibilities offered by yeasts to study the problem of the evolution of moonlighting proteins. It focuses on data available on hexokinase from Saccharomyces cerevisiae that moonlights in catabolite repression and on galactokinase from Kluyveromyces lactis that moonlights controlling the induction of the GAL genes. Possible experimental approaches to studying the evolution of moonlighting hexose kinases are suggested.

Hexokinase activity is required for recruitment of parkin to depolarized mitochondria

Autosomal recessive parkinsonism genes contribute to maintenance of mitochondrial function. Two of these, PINK1 and parkin, act in a pathway promoting autophagic removal of depolarized mitochondria. Although recruitment of parkin to mitochondria is PINK1-dependent, additional components necessary for signaling are unclear. We performed a screen for endogenous modifiers of parkin recruitment to depolarized mitochondria and identified hexokinase 2 (HK2) as a novel modifier of depolarization-induced parkin recruitment. Hexose kinase activity was required for parkin relocalization, suggesting the effects are shared among hexokinases including the brain-expressed hexokinase 1 (HK1). Knockdown of both HK1 and HK2 led to a stronger block in parkin relocalization than either isoform alone, and expression of HK2 in primary neurons promoted YFP-parkin recruitment to depolarized mitochondria. Mitochondrial parkin recruitment was attenuated with AKT inhibition, which is known to modulate HK2 activity and mitochondrial localization. We, therefore, propose that Akt-dependent recruitment of hexokinases is a required step in the recruitment of parkin prior to mitophagy.

Hexose kinases and their role in sugar-sensing and plant development.

Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms and are the origin of most of the organic matter found in nature. To be utilized, hexose sugars must first be phosphorylated. The central role of hexose-phosphorylating enzymes has attracted the attention of many researchers, leading to novel discoveries. Only two families of enzymes capable of phosphorylating glucose and fructose have been identified in plants; hexokinases (HXKs), and fructokinases (FRKs). Intensive investigations of these two families in numerous plant species have yielded a wealth of knowledge regarding the genes number, enzymatic characterization, intracellular localization, and developmental and physiological roles of several HXKs and FRKs. The emerging picture indicates that HXK and FRK enzymes found at specific intracellular locations play distinct roles in plant metabolism and development. Individual HXKs were shown for the first time to be dual-function enzymes – sensing sugar levels independent of their catalytic activity and controlling gene expression and major developmental pathways, as well as hormonal interactions. FRK, on the other hand, seems to play a central metabolic role in vascular tissues, controlling the amounts of sugars allocated for vascular development. While a clearer picture of the roles of these two types of enzymes is emerging, many questions remain unsolved, such as the specific tissues and types of cells in which these enzymes function, the roles of individual HXK and FRK genes, and how these enzymes interact with hormones in the regulation of developmental processes. It is anticipated that ongoing efforts will broaden our knowledge of these important plant enzymes and their potential uses in the modification of plant traits.

Glucose Measurement in the Presence of Tea Polyphenols

The accuracy of several commonly used methods for glucose measurement in the presence of tea polyphenols (TPLs) was investigated since TPLs (as a representative of bioactive polyphenols) and glucose often co-exist in experiments exploring TPLs’ effect on carbohydrate metabolism. The results from a model system containing only glucose and TPLs showed a TPLs’ amount-dependent variation of measured glucose concentration with a relative error (RE) of 5.0% to 35.5% when a dinitrosalicylic acid (DNS) method was used, and for glucose oxidase/peroxidase assay, the results showed a decreased content of glucose with a RE from 56.7% to 102.7%. When a hexose/kinase (HK) method was employed to quantify the glucose content, the accuracy (RE from 0.57% to 4.7%) was comparable to the result obtained by a high performance liquid chromatography (HPLC) method (RE = 0.7–3.0%) that was used as the standard control. Starch digestion experiment further demonstrated the invalidity of DNS method and the accuracy of HK method. Thus, the HK method with its accuracy and convenience is the preferred method for glucose measurement in the presence of TPLs or other bioactive polyphenols.

Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host

ABSTRACTCryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS). We sought to understand fungal carbon utilization during colonization of these fundamentally different niches within the host, in particular the roles of gluconeogenesis and glycolysis. We created mutants at key points in the gluconeogenesis/glycolysis metabolic pathways that are restricted for growth on lactate and glucose, respectively. A phosphoenolpyruvate carboxykinase mutant (the pck1∆ mutant), blocked for entry of 2- and 3-carbon substrates into gluconeogenesis and attenuated for virulence in a murine inhalation model, showed wild-type (WT) persistence in a rabbit cerebrospinal fluid (CSF) model of cryptococcosis. Conversely, both the pyruvate kinase (pyk1∆) and the hexose kinase I and II (hxk1∆/hxk2∆) mutants, which show impaired glucose utilization, exhibited severely attenuated virulence in the murine inhalation model of cryptococcosis and decreased persistence in the CNS in both the rabbit CSF and the murine inhalation models while displaying adequate persistence in the lungs of mice. These data suggest that glucose utilization is critical for virulence of C. neoformans and persistence of the yeast in the CNS.

Functional domains of yeast hexokinase 2

Hkx2 (hexokinase 2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization: it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. To get more insights into the structure–function relationships of the Hxk2 protein, we followed two different approaches. In the first, we deleted the last eight amino acids of Hxk2 and replaced Ser304 with phenylalanine to generate Hxk2wca. Analysis of this mutant demonstrated that these domains play an essential role in the catalytic activity of yeast Hxk2, but has no effect on the regulatory function of this protein. In the second, we analysed whether amino acids from Lys6 to Met15 of Hxk2 (Hxk2wrf) are essential for the regulatory role of Hxk2 and whether there is an effect on the hexose kinase activity of this protein. In the present paper, we report that the Hxk2wca mutant protein interacts with the Mig1 transcriptional repressor and the Snf1 protein kinase in the nucleus at the level of the SUC2–Mig1 repressor complex. We have demonstrated that Hxk2wca maintained full regulatory function because the glucose-repression signalling of the wild-type machinery is maintained. We also report that the Hxk2wrf mutant allele is incapable of glucose repression signalling because it does not interact with Mig1 at the level of the SUC2–Mig1 repressor complex. The two mutants, Hxk2wca and Hxk2wrf retain single functions, as a transcriptional factor or as an enzyme with hexose-phosphorylating activity, but have lost the original bifunctionality of Hxk2.

A network biology approach to aging in yeast

In this study, a reverse-engineering strategy was used to infer and analyze the structure and function of an aging and glucose repressed gene regulatory network in the budding yeast Saccharomyces cerevisiae. The method uses transcriptional perturbations to model the functional interactions between genes as a system of first-order ordinary differential equations. The resulting network model correctly identified the known interactions of key regulators in a 10-gene network from the Snf1 signaling pathway, which is required for expression of glucose-repressed genes upon calorie restriction. The majority of interactions predicted by the network model were confirmed using promoter-reporter gene fusions in gene-deletion mutants and chromatin immunoprecipitation experiments, revealing a more complex network architecture than previously appreciated. The reverse-engineered network model also predicted an unexpected role for transcriptional regulation of the SNF1 gene by hexose kinase enzyme/transcriptional repressor Hxk2, Mediator subunit Med8, and transcriptional repressor Mig1. These interactions were validated experimentally and used to design new experiments demonstrating Snf1 and its transcriptional regulators Hxk2 and Mig1 as modulators of chronological lifespan. This work demonstrates the value of using network inference methods to identify and characterize the regulators of complex phenotypes, such as aging.

Laforin Confers Cancer Resistance to Energy Deprivation–Induced Apoptosis

A long-standing but poorly understood observation in experimental cancer therapy is the heterogeneity in cancer susceptibility to energy deprivation. Here, we show that the hexose kinase inhibitor 2-deoxyglucose (2-dG) preferentially kills cancer cells with defective laforin expression and significantly increases the survival of mice with aggressive lymphoma due to a genetic defect of the laforin-encoding Epm2a gene. Normal cells from Epm2a(-/-) mice also had greatly increased susceptibility to 2-dG. Thus, laforin is a novel regulator for cellular response to energy deprivation and its defects in cancer cells may be targeted for cancer therapy.

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

Hexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete Botrytis cinerea were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Deltahxk1 mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Deltahxk1 mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. B. cinerea mutants containing hxk1 alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Deltahxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Deltahxk1 mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Deltahxk1 mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Deltaglk1 and Deltahxk1 mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, B. cinerea hexose kinases are not involved in carbon catabolite repression.

Effects of Co2+ on the erythromycin biosynthesis

Erythromycin biosynthesis is a highly complicated process, which involves both primary metabolism and secondary metabolism. The specific activities of the key enzymes related to glucose metabolism such as hexose kinase (HK), glucose-6-phosphate dehydrogenase (6-PDH), phosphofructokinase (PFK), and isocitrate dehydrogenase (ICD), were determined in Saccharopolyspora erythraea. The specific activities of the enzymes involved in secondary metabolism, such as methylmalonyl-coenzyme A mutase (MCM) and methylmalonyl-coenzyme A transcarboxylase (MCT), were detected as well. Some organic acids contained in fermentation broth were also analyzed. The results show that Co2+ is able to increase erythromycin biosynthesis. It maybe due to Co2+ improving the specific activities of methylmalonyl-coenzyme A mutase and methylmalonyl-coenzyme A transcarboxylase. Meanwhile, it also enhances the flux of the glucose metabolism pathway.

Role of tomato hexose kinases.

Hexose phosphorylation is an essential step of sugar metabolism. Only two classes of glucose and fructose phosphorylating enzymes, hexokinases (HXK) and fructokinases (FRK), have been found in plants. Tomato (Lycopersicon esculentum Mill.) is the only plant species from which four HXK and four FRK genes have been identified and characterised. One HXK and one FRK isozyme are located within plastids. The other three HXK isozymes are associated with the mitochondria, and the other three FRK isozymes are dispersed in the cytosol. These differences in location suggest that the cytoplasmic HXK and FRK have distinct roles to play in sugar metabolism. The specific roles of each of the HXK and FRK genes have been investigated using transgenic plants with modified expression of the genes. Sugar signalling effects were obtained with modified expression of the mitochondria associated HXK. In contrast, modified expression of the cytosolic FRK affected fructose metabolism rather than sugar signalling. Future research efforts will aim to determining the roles of specific hexose phosphorylating enzymes in tomato plants, the source of the hexose monomers to be phosphorylated, and their intracellular trafficking route.