Proposed mechanism and functional amino acid residues of malonyl-CoA:anthocyanin 5-O-glucoside-6'''-O-malonyltransferase from flowers of Salvia splendens,a member of the versatile plant acyltransferase family

The versatile plant acyltransferase (VPAT) family is a recently identified protein family consisting of acyltransferases involved in secondary metabolism in plants along with numerous homologues with as yet unidentified biochemical functions. Malonyl-CoA:anthocyanin 5-O-glucoside-6' "-O-malonyltransferase of Salvia splendens flowers (Ss5MaT1) is a member of this family that catalyzes the regiospecific transfer of the malonyl group from malonyl-CoA to the 6' "-hydroxyl group of the 5-glycosyl moiety of anthocyanins. To elucidate the mechanism and functional amino acid residues of VPAT family enzymes, steady-state kinetic analyses and site-directed mutagenesis of Ss5MaT1 guided by sequence comparison studies were carried out. On the basis of the results of product and dead-end inhibition studies as well as sequence comparison studies, the kinetic mechanism of Ss5MaT1 could be most consistently described in terms of a ternary complex mechanism in which both substrates and the enzyme form a complex before catalysis can occur, as in the case of chloramphenicol O-acetyltransferase (CAT) and histone acetyltransferase (HAT). Eight polar or ionizable amino acid residues that are invariant among 12 VPAT family enzymes were replaced by alanine, and the mutant enzymes were kinetically characterized. A significant diminution of the k(cat) value was observed with the substitution of His167 (relative k(cat), 0.02%) and Asp390 (<0.01%), strongly suggesting that His167 and Asp390 are very important for catalytic activity. The log k(cat) versus pH plots of the Ss5MaT1-catalyzed malonyl transfer suggested that a deprotonated active site group of pK(a) = 7.0 +/- 0.1 may be involved in the catalytic steps of the "substrate to product" conversion in the ternary enzyme-substrate complex. Taking these lines of evidence together with the suggested similarity of the kinetic and catalytic mechanisms of Ss5MaT1 to those of CAT and HAT, the following Ss5MaT1 mechanism based on general acid/base catalysis was proposed: in the ternary complex, a general base deprotonates the 6' "-hydroxyl group of the anthocyanin substrate, thereby promoting a nucleophilic attack on the carbonyl of the thioester of malonyl-CoA; His167 and Asp390 appear to be involved in the general acid/base mechanism of Ss5MaT1.     

cDNA cloning of a BAHD acyltransferase from soybean (Glycine max): isoflavone 7-O-glucoside-6'-O-malonyltransferase

A cDNA from soybean (Glycine max (L.) Merr.), GmIF7MaT, encoding malonyl-CoA:isoflavone 7-O-glucoside-6'-O-malonyltransferase, was cloned and characterized. Soybeans produce large amounts of isoflavones, which primarily accumulate in the form of their 7-O-(6'-O-malonyl-beta-D-glucosides). The cDNA was obtained by a homology-based strategy for the cDNA cloning of some flavonoid glucoside-specific malonyltransferases of the BAHD family. The expressed gene product, GmIF7MaT, efficiently catalyzed specific malonyl transfer reactions from malonyl-CoA to isoflavone 7-O-beta-D-glucosides yielding the corresponding isoflavone 7-O-(6'-O-malonyl-beta-D-glucosides) (IF7MaT activity). The k(cat) values of GmIF7MaT were much greater than those of other flavonoid glucoside-specific malonyltransferases with their preferred substrates, while the K(m) values were at comparable levels. GmIF7MaT was expressed in the roots of G. max seedlings more abundantly than in hypocotyl and cotyledon. Native IF7MaT activity was also observed in the roots, suggesting that GmIF7MaT is involved in the biosynthesis from isoflavone 7-O-beta-D-glucosides to the corresponding isoflavone 7-O-(6'-O-malonyl-beta-D-glucosides) in G. max. This protein is a member of flavonoid glucoside-specific acyltransferases in the BAHD family.     

Chicken deoxyribonuclease: purification,characterization, gene cloning and gene expression

Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn²⁺ and Mg²⁺ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu²⁺, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca²⁺ at pH 8, chicken DNase resisted to the trypsin or -mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5-untranslated region and 166 of the 3 and, in the 5-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 ± 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.     

Dimalonated anthocyanins from the flowers of Salvia splendens and S. coccinea

The pigments of Salvia splendens flowers (scarlet cvs) have been identified as pelargonidin 3-caffeoylglucoside-5-dimalonylglucoside and pelargonidin 3-p-coumaroylglucoside-5-dimalonylglucoside. The flowers of S. coccinea contain the same pigments in the corolla but the calyx contains, in addition, the cyanidin analogues.     

cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora

Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less understood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) ( EC 2.3.1.153 ) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52 736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multifunctional acyltransferases ( St-Pierre et al . (1998 ) Plant J. 14, 703–713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.     

粘质沙雷氏菌H3010发酵型D-乳酸脱氢酶基因的克隆表达、纯化及酶学性质研究

通过PCR技术从粘质沙雷氏菌H3010基因组DNA中扩增出该D-乳酸脱氢酶基因,连接至pET-28a（＋）表达载体,转入大肠杆菌BL21（DE3）中进行了重组表达,优化了酶纯化的条件,并对其酶学性质进行初步研究。结果表明,获得的该酶编码基因全长993bp,编码330个氨基酸,大小为37kDa。经优化表达及纯化条件后重组酶纯度可达90％。酶学性质研究发现,该重组酶最适反应温度为60℃,最适酶促反应pH为7．5（O．2mol／L磷酸盐缓冲液）,37℃下测得对底物丙酮酸的动力学参数Km=3．39mmol／L,Vmax=6．87mmol／（mg·min）,对辅酶NADH的动力学参数Km=1．43mmol／L,Vmax=1．61mmo]／（mg·min）。为酶法生产D-乳酸及利用代谢工程构建产D-乳酸的基因工程菌打下基础。     

Molecular cloning, gene expression, purification and characterization of fermentative D-lactate dehydrogenase from S.marcescens H3010

通过PCR技术从粘质沙雷氏菌H3010基因组DNA中扩增出该D-乳酸脱氢酶基因,连接至pET-28a(+)表达载体,转入大肠杆菌BL21 (DE3)中进行了重组表达,优化了酶纯化的条件,并对其酶学性质进行初步研究.结果表明,获得的该酶编码基因全长993 bp,编码330个氨基酸,大小为37 kDa.经优化表达及纯化条件后重组酶纯度可达90％.酶学性质研究发现,该重组酶最适反应温度为60℃,最适酶促反应pH为7.5(0.2 mol/L磷酸盐缓冲液),37℃下测得对底物丙酮酸的动力学参数Km =3.39 mmol/L,Vmax =6.87 mmol/( mg · min),对辅酶NADH的动力学参数Km=1.43 mmol/L,Vmax=1.61 mmol/( mg· min).为酶法生产D-乳酸及利用代谢工程构建产D-乳酸的基因工程菌打下基础.     

A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.     

An extracellular exo-beta-(1,3)-glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression

An extracellular exo-beta-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 micromole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; pI, 4.46) that shares 36-64% amino acid identity with other yeast exo-beta-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.     

The phenylalanine ammonia-lyase gene family in Salvia miltiorrhiza: genome-wide characterization, molecular cloning and expression analysis

Salvia miltiorrhiza Bunge is a well-known material of traditional Chinese medicine. Hydrophilic phenolic acids, such as rosmarinic acid and salvianolic acid B, are a group of pharmaceutically important compounds in S. miltiorrhiza. The biosynthesis of rosmarinic acid requires the coordination of the phenylpropanoid pathway and the tyrosine-derived pathway. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. Systematic analysis of the SmPAL gene family has not been carried out. We report here the identification of three SmPALs through searching the recently obtained working draft of the S. miltiorrhiza genome and full-length cDNA cloning. Bioinformatic and phylogenetic analyses showed that SmPAL1 and SmPAL3 clustered in a sub-clade of dicot PALs, whereas SmPAL2 fell into the other one. Some important cis-elements were conserved in three SmPAL promoters, whereas the others were not. SmPAL1 and SmPAL3 were highly expressed in roots and leaves of S. miltiorrhiza, but SmPAL2 were predominately expressed in stems and flowers. It indicates that SmPAL1 and SmPAL3 function redundantly in rosmarinic acid biosynthesis. All SmPALs were induced in roots treated with PEG and MeJA, but the time and degree of responses were different, suggesting the complexity of SmPAL-associated metabolic network in S. miltiorrhiza. This is the first comprehensive study dedicated to SmPAL gene family characterization. The results provide a basis for elucidating the role of SmPAL genes in the biosynthesis of bioactive compounds.     

Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1: purification,characterization, gene cloning,and heterologous expression

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k(cat), 5.41 s(-1); K(m), 32 micro M) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k(cat), 351 s(-1); K(m), 214 micro M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.     

Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.

6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.     

Proline dehydrogenase from Pseudomonas fluorescence: gene cloning, purification, characterization and homology modeling

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescence was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K(m) and V(max) values of the P. fluorescence ProDH were estimated to be 35 mM and 116 micromol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30 degrees C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescence ProDH.     

Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning

The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.     

Biosynthesis of monoterpenes. Partial purfication and characterization of a bicyclic monoterpenol dehydrogenase from sage (Salvia officinalis)

Galactose 1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose 1-phosphate uridylyltransferase, EC 2.7.7.12) was isolated from human red cells by DEAE-cellulose and hydroxylapatite chromatography. The enzyme consists. of two similar subunits of molecular weight 44,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was found to be 67,000 by Sephadex G-200 chromatography and 88,000 by ultracentrifugation studies in sucrose density gradients. The specific activity of the purified enzyme was about 40 μmoles per min per mg of protein.