Analysis of the Escherichia coli glucosamine-6-phosphate synthase activity by isothermal titration calorimetry and differential scanning calorimetry

Glucosamine-6-phosphate synthase (GlmS) is responsible for the first and rate-limiting step in the hexosamine biosynthetic pathway. It catalyzes the conversion of D-fructose-6P (F6P) into D-glucosamine-6P (GlcN6P) using L-glutamine (Gln) as nitrogen donor (synthase activity) according to an ordered bi-bi process where F6P binds first. In the absence of F6P, the enzyme exhibits a weak hydrolyzing activity of Gln into Glu and ammonia (glutaminase activity), whereas the presence of F6P strongly stimulates it (hemi-synthase activity). Until now, these different activities were indirectly measured using either coupled enzyme or colorimetric methods. In this work, we have developed a direct assay monitoring the heat released by the reaction. Isothermal titration calorimetry and differential scanning calorimetry were used to determine kinetic and thermodynamic parameters of GlmS. The direct determination at 37 °C of kinetic parameters and affinity constants for both F6P and Gln demonstrated that part of the ammonia produced by Gln hydrolysis in the presence of both substrates is not used for the formation of the GlcN6P. The full characterization of this phenomenon allowed to identify experimental conditions where this leak of ammonia is negligible. Enthalpy measurements at 25 °C in buffers of various heats of protonation demonstrated that no proton exchange with the medium occurred during the enzyme-catalyzed glutaminase or synthase reaction suggesting for the first time that both products are released as a globally neutral pair composed by the Glu carboxylic side chain and the GlcN6P amine function. Finally we showed that the oligomerization state of GlmS is concentration-dependent.     

Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase

A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.     

Phosphatidylinositol synthase of Tetrahymena: inositol isomers as substrates in phosphatidylinositol biosynthesis and headgroup exchange reactions

Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Solubilization of rhamnogalacturonan I galactosyltransfrases from membranes of a flax cell suspension

Galactosyltransferases (GalTs), capable of transferring a galactosyl residue from UDP-galactose (UDP-Gal) to polysaccharide acceptor, were solubilized from flax (Linum usitatissimum L.) membranes using 0.5% CHAPS. The observed requirement for a rhamnogalacturonan I (RG-I) exogenous substrate to stimulate the solubilized GalT activity provided the first evidence for the presence of RG-I GalT activities in flax cells. An assay to measure specifically the products of this RG-I GalT activity was designed, based on size-exclusion chromatography. Labelled products were characterized as an RG-I polymer by using purified RG-I hydrolase or lyase. At pH 8 and in the presence of 5 mM CaCl2, beta-D-galactosyl residues were specifically transferred onto RG-I branches of short beta-(1 --> 4)-D-galactan side chains. These side chains were liable to hydrolysis by beta-galactosidase and endo-beta-(1 --> 4)-D-galactanase. The RG-I GalT had a temperature optimum of 30 degrees C. an apparent Km for UDP-Gal and exogenous RG-I substrate of 460 +/- 40 microM and 1.1 +/- 0.1 mg ml(-1) respectively, and a Vmax of 3.0 +/- 0.5 pkat mg(-1) protein.     

Glycosyltransferase mechanisms: impact of a 5-fluoro substituent in acceptor and donor substrates on catalysis

In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and beta-N-acetylglucosaminyl-beta-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc beta-octyl glycoside acceptor, however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.     

Potential of a 7-dimethylallyltryptophan synthase as a tool for production of prenylated indole derivatives

Recently, a gene for a 7-dimethylallyltryptophan synthase (7-DMATS) was identified in Aspergillus fumigatus and its enzymatic function was proven biochemically. In this study, the behaviour of 7-DMATS towards aromatic substrates was investigated and compared with that of the 4-dimethylallyltryptophan synthase FgaPT2 from the same fungus. In total, 24 simple indole derivatives were tested as potential substrates for 7-DMATS. With an exception of 7-methyltryptophan, all of the substances were accepted by 7-DMATS and converted to their prenylated derivatives, indicating a more flexible substrate specificity of 7-DMATS in comparison to that of FgaPT2. The relative activities of 7-DMATS towards these substrates were from 4% to 89% of that of L-tryptophan, much higher than that of FgaPT2. Structural elucidation of the isolated enzymatic products by nuclear magnetic resonance and mass spectrometry analysis proved unequivocally the prenylation at position C7 of the indole ring. Overnight incubation with eight substances showed that the conversion ratios were in the range of 55.9% to 99.7%. This study provided an additional example that prenylated indole derivatives can be effectively produced by using the overproduced and purified 7-DMATS.     

Co-translational function of Cosmc, core 1 synthase specific molecular chaperone, revealed by a cell-free translation system

The core 1 structure of the mucin type O-glycan is synthesized by core 1 β1,3-galactosyltransferase (C1GalT). Core 1 synthase specific molecular chaperone (Cosmc), a molecular chaperone specific for C1GalT, is essential for the expression of functional C1GalT in mammalian cells. In this study, we have established a procedure for detecting the chaperone activity of Cosmc by using a wheat germ cell-free translation system. Active C1GalT was expressed following simultaneous translation with Cosmc or translation in the presence of recombinant Cosmc protein. Moreover, we show that recombinant Cosmc must be present during the translation of C1GalT, as it is ineffective when added after translation. These results indicate that Cosmc mediates the co-translational activation of C1GalT and that it may prevent the unfavorable aggregation of C1GalT.     

Quantitative continuous assay for hyaluronan synthase

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.     

Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity

We report on the cDNA cloning and characterization of a novel short-chain isoprenyl diphosphate synthase from the aphid Myzus persicae. Of the three IPPS cDNAs we cloned, two yielded prenyltransferase activity following expression in Escherichia coli; these cDNAs encode identical proteins except for the presence, in one of them, of an N-terminal mitochondrial targeting peptide. Although the aphid enzyme was predicted to be a farnesyl diphosphate synthase by BLASTP analysis, rMpIPPS, when isopentenyl diphosphate and dimethylallyl diphosphate are supplied as substrates, typically generated geranyl diphosphate (C10) as its main product, along with significant quantities of farnesyl diphosphate (C15). Analysis of an MpIPPS homology model pointed to substitutions that could confer GPP/FPP synthase activity to the aphid enzyme.     

Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli

Flavonols, a class of bioactive polyphenols present in plants, are the products of flavonol desaturation catalyzed by flavonol synthase (FLS). We cloned the cDNA coding for the enzyme FLS from Camellia sinensis (CsFLS) by end-to-end PCR followed by 5'- and 3'-RACE. The putative CsFLS had 333 amino acid residues, displayed identities to the FLSs of Arabidopsis and Ginkgo of 53% and 52.5%, respectively, and contained several conserved elements found in the 2-oxoglutarate-Fe(II)-dioxygenase superfamily. The cDNA of CsFLS was subcloned into pET28a(+) and introduced into Escherichia coli (BL21-CodonPlus-RIL). Induction with 0.1mM IPTG at low temperature (20 degrees C) led to higher amounts of CsFLS in the soluble fraction than induction at 30 degrees C. The enzyme aggregated into inclusion bodies could be rescued by denaturation with 6M urea and purification with a His.Bind purification kit. The purified protein was desalted by Amicon Ultra-15 centrifugal filter unit, and the His-tag was removed with thrombin. The finally purified protein was assayed with dihydroquercetin as substrate and the products were analyzed by HPLC. The addition of FeSO(4) to the buffers used in the CsFLS purification significantly increased the recovery of active enzyme. The CsFLS obtained in this study was found to have higher specific activity and lower K(m) than previously reported FLSs.     

Distinct substrate specificities and unusual substrate flexibilities of two hydroxycinnamoyltransferases,rosmarinic acid synthase and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl-transferase,from <Emphasis Type="Italic">Coleus blumei</Emphasis> Benth.

cDNAs and genes encoding a hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS; rosmarinic acid synthase) and a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CbHST) were isolated from Coleus blumei Benth. (syn. Solenostemon scutellarioides (L.) Codd; Lamiaceae). The proteins were expressed in E. coli and the substrate specificity of both enzymes was tested. CbRAS accepted several CoA-activated phenylpropenoic acids as donor substrates and d-(hydroxy)phenyllactates as acceptors resulting in ester formation while shikimate and quinate were not accepted. Unexpectedly, amino acids (d-phenylalanine, d-tyrosine, d-DOPA) also yielded products, showing that RAS can putatively catalyze amide formation. CbHST was able to transfer cinnamic, 4-coumaric, caffeic, ferulic as well as sinapic acid from CoA to shikimate but not to quinate or acceptor substrates utilized by CbRAS. In addition, 3-hydroxyanthranilate, 3-hydroxybenzoate and 2,3-dihydroxybenzoate were used as acceptor substrates. The reaction product with 3-aminobenzoate putatively is an amide. For both enzymes, structural requirements for donor and acceptor substrates were deduced. The acceptance of unusual acceptor substrates by CbRAS and CbHST resulted in the formation of novel compounds. The rather relaxed substrate as well as reaction specificity of both hydroxycinnamoyltransferases opens up possibilities for the evolution of novel enzymes forming novel secondary metabolites in plants and for the in vitro formation of new compounds with putatively interesting biological activities.     

虎杖苯亚甲基丙酮合酶PcPKS2的表达纯化和晶体生长

植物类型Ⅲ聚酮合酶超家族(PKSs),又称查尔酮合酶(Chalcone synthase,CHS)超家族,催化合成多种植物次生代谢产物的分子骨架。苯亚甲基丙酮合酶(Benzalacetone synthase,BAS)催化4-香豆酰辅酶A与丙二酰辅酶A通过一步脱羧缩合反应生成苯亚甲基丙酮,是一系列具有重要生物学活性苯丁烷类化合物及其衍生物的前体化合物。前期工作从虎杖中分离出苯亚甲基丙酮合酶BAS(PcPKS2)和1个具有CHS和BAS活性的双功能酶(PcPKS1)。两者与超家族其他成员序列经比较,在包括门卫氨基酸Phe215和Phe265在内的重要氨基酸序列存在一定差异。已有蛋白晶体学研究结果表明,PKSs家族不同成员的功能多样性来自于酶催化位点的非常微小的构象变化。为了能够从结构上比较PcPKS2和Pc PKS1双功能酶活性差异可能产生的机制,以确定其高效BAS活性的分子机理,研究利用了大肠杆菌原核表达系统过量表达了C-端融合有His6标签的重组蛋白,经纯化得到了高纯度蛋白。经过对其晶体生长条件进行摸索和优化,得到了能用于X-射线衍射的单晶,为其结构解析、催化机理研究、了解虎杖聚酮类化合物生物合成机制和该类酶在基因工程中的应用提供了基础。     

Exploring biosynthetic diversity with trichodiene synthase

Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP(i)). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.85A resolution X-ray crystal structure of the complex with Mg(2+) (3)-PP(i) and the benzyl triethylammonium cation, which is a bulkier mimic of the bisabolyl carbocation intermediate in catalysis. Taken together, these findings show that the active site of trichodiene synthase is sufficiently flexible to accommodate bulkier and electronically-diverse substrates and intermediates, which could indicate additional potential for the biosynthetic utility of this terpenoid cyclase.     

Sucrose phosphate synthase and other sucrose metabolizing enzymes in fruits of various species

Recent reports have suggested that sucrose phosphate synthase (EC 2.4.1.14), a key enzyme in sucrose biosynthesis in photosynthetic “source” tissues, may also be important in some sucrose accumulating “sink” tissues. These experiments were conducted to determine if sucrose phosphate synthase is involved in sucrose accumulation in fruits of several species. Peach (Prunus persica NCT 516) and strawberry (Fragaria x ananassa cv. Chandler) fruits were harvested directly from the plant at various stages of fruit development. Kiwi (Actinidia chinensis), papaya (Carica papaya), pineapple (Ananas comosus) and mango (Mangifera indica) were sampled in postharvest storage over a period of several days. Carbohydrate concentrations and activities of sucrose phosphate synthase, sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. All fruits contained significant activities of sucrose phosphate synthase. Moreover, in fruits from all species except pineapple and papaya, there was an increase in sucrose phosphate synthase activity associated with the accumulation of sucrose in situ. The increase in sucrose concentration in peaches was also associated with an increase in sucrose synthase activity and, in strawberries, with increased activity of both sucrose synthase and neutral invertase. The hexose pools in all fruits were comprised of equimolar concentrations of fructose and glucose, except in the mango. In mango, the fructose to glucose ratio increased from 2 to 41 during ripening as sucrose concentration more than doubled. The results of this study indicate that activities of the sucrose metabolizing enzymes, including sucrose phosphate synthase, within the fruit itself, are important in determining the soluble sugar content of fruits of many species. This appears to be true for fruits which sweeten from a starch reserve and in fruits from sorbitol translocating species, raffinose saccharide translocating species, and sucrose translocating species.     

Ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in Bacteria and Archaea,which contain inositol phospholipid

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes.     

Purification and properties of glutamate synthase from Streptomyces lincolnensis

Glutamate synthase (EC 1 4 1 14) was purified to homogeneity from a cell\|free extract of Streptomyces lincolnensis by precipitation with streptomycin sulfate and ammonium sulfate, and column chromatography on DEAE\| cellulose, Sepharose 6B, DEAE\|sephadex A\|50, hydroxyapatite and Sephadex G\|150. The enzyme activity is stabilized by addition of α ketoglutarate, PMSF,EDTA, β mercaptoethanol and glycerol. The native enzyme has a molecular weight of 138 000 and is composed of two nonidentical subunits with molecular weights of 81 000 and 57 000. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The enzyme shows optimum activity at pH 7.2 and 30℃. Km values for α ketoglutarate, L\|glutamine and NADH were 417, 435, and 52.1 μmol/L, respectively. When NADPH was substituted for NADH as reductant, there was approximately 13% of the control activity. The activity of this glutamate synthase is inhibited by its products (i.e. glutamate and NAD), several metal ions, amino acids and tricarboxylic acid cycle intermediates.     

Artifactual detection of ADP-dependent sucrose synthase in crude plant extracts.

Results presented in a previous report from this laboratory indicated the presence, in crude extracts from sycamore (Acer pseudoplatanus) and spinach (Spinacea oleracea), of a sucrose synthase (EC 2.4.1.13) showing high affinity for ADP as the glucose acceptor in the sucrose-cleaving reaction. In the present paper we report that the modified enzymatic method previously used to measure sucrose synthase activities leads to the detection of artifactual ADP-dependent sucrose synthase, which in fact arises from the combined action of invertase (EC 3.2.1.26) and nucleoside diphosphate kinase (EC 2.7.4.6) activities. We also present data on the partial purification of nucleoside diphosphate kinase from sycamore cells.